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BOR - Papers in Press, published online ahead of print May 28, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.016535
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BIOLOGY OF REPRODUCTION 69, 1201–1207 (2003)
DOI: 10.1095/biolreprod.103.016535
© 2003 by the Society for the Study of Reproduction, Inc.

Testis

Does Alligator Testis Produce Estradiol? A Comparison of Ovarian and Testicular Aromatase1

Valentine A. Lance2,3, Alan J. Conley4, Samantha Mapes4, Colin Steven5, and Allen R. Place5

Center for Reproduction of Endangered Species,3 San Diego, California 92112 Department of Population Health and Reproduction,4 School of Veterinary Medicine, University of California, Davis, California 95616 Center of Marine Biotechnology,5 Maryland Biotechnology Institute, University of Maryland, Baltimore, Maryland 21202


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Testicular secretion of estradiol is necessary for normal spermatogenesis and male reproductive physiology in humans and rodents. The role of estradiol in nonmammalian vertebrates remains unknown, but elevated circulating estradiol has been reported in male lizards, alligators, and various bird species. We have been unable to detect circulating estradiol in male alligators; therefore, we reexamined the question of testicular production of estradiol in alligators using more rigorous assay procedures. A large pool of plasma from a male alligator was extracted and run through an HPLC column. Immunoreactive estradiol-like material eluted coincident with authentic estradiol. By using an ultrasensitive RIA and processing large volumes of male plasma (1000 µl), we were able to measure estradiol. Estradiol in male alligators ranged from 0.23 to 3.14 pg/ml, whereas estradiol in immature female alligators ranged from 14 to 66 pg/ml. Aromatase activity in microsomes from adult alligator ovarian tissue was 36.2 ± 1.6 pmol mg-1 h-1, whereas activity in testicular microsomes ranged between 0.92 and 2.38 pmol mg-1 h-1. Ovarian aromatase activity was inhibited in a concentration-dependent fashion by Fadrozole, but the essentially background activity of testicular aromatase was not inhibited at any concentration of Fadrozole. Likewise, a comparison of alligator testicular and ovarian aromatase mRNA expression gave a similar result: the ovarian expression was 600-fold higher and brain tissue was 10-fold higher than that of the testis. Circulating estradiol in male alligators is probably of extragonadal origin, and the testis produces little if any of this steroid.

estradiol, male reproductive tract, steroid hormones, testis, testosterone


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The role of estrogen in male reproductive function remains unclear. In estrogen-deficient mice, spermatogenesis is seriously impaired [1–4], whereas in the few known cases of estrogen deficiency in human males the data are ambiguous [4, 5]. The Leydig cells of humans [6] and several other mammalian species [7] express aromatase and secrete estradiol in vitro. Rat testicular germ cells also express aromatase [8]. In a human male with a mutation in the estrogen receptor (ER-{alpha}) who was thus estrogen resistant, serum estradiol was elevated and LH was elevated, suggesting that testicular estrogen secretion plays a negative feedback role in pituitary control of gonadotropin secretion [9]. In nonmammalian vertebrates, the role of estrogen in male reproductive physiology is even less clear. Estrogen receptors have been identified in the male reproductive tract of birds [10] and a lizard [11], and a role for estradiol in lizard spermatogonial proliferation has been suggested [12]. Aromatase mRNA is expressed in the testis of adult chickens [13], but could not be detected in the testes of adult zebra finches [14]. Neonate male zebra finches have elevated serum estradiol that is believed to be of extragonadal origin [15, 16], whereas adult male zebra finches have little circulating estradiol [14]. Circulating estradiol levels in the 20–80 pg/ml range have been reported in males of various bird species [17–20]. Males of the lizard Podarcis sicula exhibit extremely high circulating plasma levels of estradiol, >1000 pg/ml [21]. Following treatment with an aromatase inhibitor, circulating estradiol in these male lizards was reduced by about 90% [22]. In male alligators, circulating estradiol levels are usually too low to be detected by most assays. Plasma estradiol in preovulatory female alligators ranges from 200 to 1200 pg/ml [23–26] and in neonate female alligators is in the range of 20–30 pg/ml, but plasma estradiol is undetectable in neonate males and juvenile and adult males [27] (V. Lance, unpublished data). In Florida, however, juvenile male alligators have been reported to have circulating estradiol concentrations ranging from 10 to 60 pg/ml [28–33]. Given this discrepancy in results, we reexamined circulating estradiol and aromatase activity in male alligators.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Alligator blood samples were collected throughout the year from juvenile and adult male and female alligators at the Rockefeller Wildlife Refuge in Louisiana as part of an ongoing study on growth and sexual maturation of the species (Lance and Elsey, unpublished results). Twenty-five blood samples from adult male alligators (>1.85 m total length) and 24 blood samples from juvenile male alligators (<1.85 m total length) were assayed (Table 1). Fifteen blood samples from juvenile female alligators (0.89–1.36 m total length) also were assayed for estradiol. Testicular and ovarian tissues were collected from nuisance animals and from animals taken for other unrelated research. Three adult male alligators, total body length 198, 257, and 264 cm, and two juvenile male alligators with total body lengths of 152 and 156 cm were used for the aromatase assays. The three ovarian samples came from reproductively active animals undergoing vitellogenesis. The three female alligators ranged in size from 227 to 247 cm total length. Follicular diameter ranged from 21 to 25 mm. Preovulatory follicular diameter in mid to late May is close to 40 mm [23]. Yolk was expressed from three ovarian follicles from each animal, and the tissue was frozen immediately on dry ice. Testis tissue was collected from immature and mature alligators and frozen immediately on dry ice. The five male testis samples came from alligators collected in March and April, when testosterone is elevated and spermatogenesis is approaching peak activity [23]. Animal procedures were performed according to guidelines and under the supervision of the Louisiana Fish and Wildlife Department.


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  		TABLE 1. Plasma estradiol and testosterone in alligators

Radioimmunoassay

Plasma estradiol-17ß was measured using an [125I]-estradiol RIA kit (Diagnostics Products, Los Angels, CA) that was modified to increase the sensitivity of the assay. Modifications included using extracted rather than unextracted plasma, reconstituting the dried extract in PBS buffer, pH 7.0, using only 50 µl of primary antibody instead of the recommended 100 µl, and using dextran-charcoal instead of the second antibody to separate bound from free steroid. A series of standards from 0.5 to 100 pg in PBS buffer were used rather than the human serum standards that come with the kit. With these modifications, a sensitivity of <0.5 pg/assay tube was achieved [27]. An extracted buffer sample gave a reading of 0.08 pg/ml. Cross-reactivity of estrone and estriol against the antibody was 12.5% and 0.24%, respectively. Duplicate plasma samples (1000 µl) were extracted with 4 ml ethyl acetate:n-hexane (3:2), and the extract was dried under a stream of air at 37°C and reconstituted in 200 µl assay buffer. Antibody (50 µl) was added, the tubes were vortexed briefly and then incubated for 2 h at room temperature, 100 µl [125I]-estradiol was added, and the tubes were vortexed again and incubated for 1 h at room temperature. Charcoal:dextran suspension (0.5 ml) was added, and the tubes were incubated for 15 min at room temperature. The tubes were then centrifuged for 15 min at 3000 x g. From each tube, 600 µl of supernatant was carefully pipetted into 12- x 75-mm glass culture tubes, and the radioactivity was measured in a gamma counter.

Testosterone was assayed as described previously [34]. For both estradiol and testosterone, quality control standards consisting of a pool of alligator plasma and commercial controls of pooled human serum of known hormone concentrations (Diagnostics Products) were run with every assay.

Interassay coefficients of variation were 8.4% for estradiol and 10.5% for testosterone. Intraassay variation was 3% for estradiol and 5% for testosterone.

High Performance Liquid Chromatography

Approximately 100 ml of blood from a large adult male alligator (302 cm total length) was taken from the dorsal sinus using an 18-ga, 36-mm needle and a 50-ml syringe. The alligator was then released. The plasma was extracted in batches with ethyl acetate:hexane (3:2), and the extracts were combined and dried. The extract was taken up in water, extracted using a solid phase cartridge (Prep-sep; Supelco, Bellefonte, PA), and eluted with methanol, and the eluate was dried. Estradiol (2,4,6,7-3H (N); Perkin Elmer, Boston, MA) in 10 µl methanol was added to the dried eluate, and an additional 15 µl of methanol was added. The tube was vortexed, and 20 µl of the methanolic solution was injected into the HPLC column.

HPLC analysis was performed using isocratic elution on a Waters (Milford, MA) model 501 with an Xterra MS C18 5-µm column. The mobile phase consisted of a 0.005 M phosphate buffer with methanol:water (3:2) adjusted to pH 4.5 at a flow rate of 0.75 ml/min. The eluate was split such that 200 µl of each milliliter went directly to a ß-RAM Model 3 radiochromatography detector (IN/US Systems, Tampa, FL) for tritium detection, and the remainder was directed to a fraction collector where 1-ml fractions were collected up to a total of 90 tubes. The fractions were dried in a Savant Speed-Vac (Savant Instruments, Holbrook, NY), reconstituted in 0.2 ml of estradiol assay buffer, and directly assayed for estradiol using the modified [125I]-estradiol kit. Using this system, fractions could be assayed for estradiol without interference from the tritium-labeled internal marker.

RNA Extraction

Total RNA was extracted from tissue samples (testes, n = 3; ovaries, n = 3; brain, n = 3; oviduct, n = 3; kidneys, n = 6; liver, n = 6) of six alligators (three males, three females) using a FastRNA Kit (Bio101, Carlsbad, CA) and stored at -80°C. A portion (200 mg) of each frozen sample was added to homogenization tubes with 1 ml Trizol reagent. Samples were homogenized for 20 sec at a setting of 6.0 in a FastPrep FP120 and incubated on ice for 5 min. Chloroform (200 µl) was added to each sample and mixed by inversion for 20 sec, followed by a 3-min incubation at room temperature. The phases were separated by centrifugation (12 000 rpm for 15 min at 4°C), and the aqueous phase was removed to a clean tube. Isopropanol (500 µl) was added to each of the samples, which were then mixed by inversion and incubated for 10 min at room temperature. RNA was pelleted by centrifugation (12 000 rpm for 10 min at 4°C), washed with 1 ml of ice-cold 75% ethanol, air dried, and resuspended in 100 µl Rnase-free distilled water.

First-Strand cDNA Synthesis

Complementary DNA was synthesized using total RNA isolated from the frozen alligator tissues. Two micrograms of total RNA was mixed with 2.0 µl of 0.5 µg/µl random hexamer and water to a total volume of 12 µl. These mixtures were heated to 70°C for 5 min, and 13 µl of the master mix (5.0 µl 5x Moloney murine leukemia virus reverse transcription [MMLV-RT] buffer, 5.0 µl 10 mM dNTP, 0.5 µl RNasin, and 1.0 µl MMLV-RT) was added. These reactions were incubated with the following temperature regimes: 25°C for 5 min, 37°C for 30 min, 42°C for 30 min, and 75°C for 5 min, after which they were transferred to clean tubes and stored on ice.

Quantitative Kinetic Reverse Transcription Polymerase Chain Reaction for Aromatase and ß-Actin mRNA

The relative mRNA levels of aromatase and ß-actin were quantified in the RNA samples using a fluorescence-based quantitative real-time polymerase chain reaction (PCR) assay as previously described [35]. Samples were run on an ABI Prism 7700 Sequence Detector (Applied Biosystems, Foster City, CA) in triplicate using 20 ng of cDNA as template and using gene-specific primers for alligator aromatase (forward, 5'-TGCAGAGCACCCTAAGGTAGAAG-3'; reverse, 5'-TGGCTGGTATCTCATGCTCTCA-3') and alligator ß-actin (forward, 5'-GTGATCACCATCGGCAATGA-3'; reverse, 5'-GGAGTTGAAAGTGGTCTCGTGAAT-3') and SybrGreen (Applied Biosystems, Foster City, CA) as the indicator. Each reaction was set up as follows: 2.5 µl 10x buffer, 3.0 µl 25 mM MgCl2, 2.0 µl 12.5 mM dNTP, 0.125 µl AmpliTaq Gold, 0.25 µl AmpErase, 1.5 µl 5 µM forward primer, 1.5 µl 5 µM reverse primer, 5 µl 4 ng/µl cDNA, and 9.1 µl distilled water for a total volume of 25 µl. PCR conditions were 50°C for 2 min, 95°C for 10 min, and then 50 cycles of 95°C for 15 sec and 60°C for 1 min.

Aromatase Assays

Subcellular fractionation Testicular and ovarian tissue (0.5 g) was homogenized in a glass hand-held homogenizer in 1 ml phosphate buffer (0.1 M KPO4, pH 7.4, 20% glycerol, 5 mM ß-mercaptoethanol, 0.5 mM phenylmethane sulonylfluoride (PMSF), and 0.1 µg/ml aprotinin) per 0.1 g tissue. Samples were sonicated for 3 sec and then centrifuged for 10 min at 15 000 x g. The supernatant was subsequently centrifuged for 1 h at 100 000 x g. The resulting microsomal pellet was resuspended in 0.5 ml of phosphate buffer with 1 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate using a hand-held homogenizer and sonicated for 1 sec. A bicinchoninic acid protein assay (Pierce, Rockford, IL) was used to determine protein concentration, and the individual microsomal preparations were stored in aliquots at -80°C.

Tritiated water assay Aromatase activity in the microsomal fractions was estimated by measuring the incorporation of tritium from [1ß-3H]-androstenedione (New England Nuclear, Wilmington, DE) into 3H2O as validated by Corbin et al. [36, 37]. Incubations containing 300 nM androstenedione (10% labeled, 90% radio inert; Steraloids, Wilton, NH) were conducted in duplicate at 33°C for 2 h in the presence or absence of aromatase inhibitor Fadrazole (4-(5,6,7,8-tetrahydroimidazo [1,5a]pyridin-5yl) benzonitrile monohydrochloride, CGS 16949; Ciba Geigy, Summit, NJ) at final concentrations ranging from 0.03 to 333 nM in 0.5 ml 50 mM phosphate buffer with 1 mM EDTA. Control tubes received equivalent amounts of ethanol not to exceed 1%. Microsomes (50 µg) were incubated with the substrate in the presence of an NADPH-generating system containing 1 mM NADPH, 2 mM NADP, 17 mM glucose-6-phosphate, and 1 unit of glucose-6-phosphate dehydrogenase (Sigma Chemical Co., St Louis, MO). The reactions were stopped with 0.25 ml cold 30% trichloroacetic acid. Samples were extracted subsequently with 1 ml chloroform and centrifuged for 10 min at 2000 x g. The aqueous phase (0.5 ml) was removed and combined with 0.5 ml of 5% charcoal and 0.5% dextran suspension and centrifuged for 30 min at 2000 x g. An aliquot (0.5 ml) of the supernatant was removed, and the activity was quantified by liquid scintillation counting. Background values for tritium recovered from tubes with all assay components except microsomal protein were subtracted from each sample. Verification of estrone product formation by HPLC and thin-layer chromatography has been reported previously [36, 37]. Activity was expressed per milligram of microsomal protein.

Statistical Analysis

The means of hormone concentrations for juvenile and adult alligators were compared using an unpaired t-test, and simple linear regression analysis was used to analyze the relationship between testosterone and estradiol.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
HPLC and RIA

From the extract of the pool of plasma from a large male alligator, a peak of immunoreactive estradiol-like material was eluted in the same position as authentic tritium-labeled estradiol. Two small peaks of immunoreactive material were eluted just before the estradiol peak, but the identity of the material could not be determined with certainty (Fig. 1). In this system, estrone runs at the approximate position of the larger of the two small peaks.



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  		FIG. 1. Elution profile of authentic 3H-labeled estradiol ({square}) and immunoreactive estradiol ({blacktriangleup}) from an extract of 100 ml of male alligator plasma. Radioactivity is expressed as counts/min (cpm), and immunoreactivity is expressed as pg/ml

Circulating estradiol was detected in male alligators but in very low concentrations. Only when 1000 µl of plasma was extracted and assayed using the ultrasensitive [125I]-estradiol kit could estradiol be reliably quantified. Plasma estradiol levels of 0.29–3.14 pg/ml were determined for a series of both adult and immature males. Results from adult male alligators (>1.85 m total length) and immature male alligators (<1.85 m) are given in Table 1. There was a highly significant correlation between testosterone and estradiol in the blood samples from adult male alligators (r2 = 0.904, P = 0.0001; Fig. 2A), but there was no significant correlation between testosterone and estradiol in the immature alligators (r2 = 0.002, P = 0.838) (see Fig. 2B). Because some of the adult male samples had extremely high testosterone levels (40–100 ng/ml), we tested the effect of high concentrations of testosterone in the estradiol assay. Although the manufacturers of the estradiol kit reported no detectable cross-reactivity for either testosterone or 5{alpha}-dihydrotestosterone against the antibody, when we tested 100, 50, and 20 ng of testosterone in the assay values of 2.1, 1.0, and 0.4 pg/ml were recorded as estradiol. Therefore, the values of estradiol in samples with testosterone concentrations >10 ng/ml were adjusted to account for this cross-reactivity.



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  		FIG. 2. Plot of plasma testosterone (ng/ml, y-axis) and plasma estradiol (pg/ml, x-axis) in adult male alligators (A) and juvenile male alligators (B). Note the differences in scale for testosterone values between adults (0–150 ng) and juveniles (0–20 ng)

Estradiol levels in immature male alligators were significantly higher than those in adult alligators (P = 0.0003; immature: 1.53 ± 0.16 pg/ml, n = 24; mature: 0.85 ± 0.08 pg/ml, n = 27). There was also a significant difference between spring (March to May) and summer (June to September) in both the adult males (spring: 1.15 ± 0.09 pg/ml; summer: 0.62 ± 0.08 pg/ml; P = 0.001) and immature males (spring: 1.90 + 0.21 pg/ml; summer: 1.08 ± 0.19 pg/ml; P = 0.009). Estradiol levels in a series of immature females ranged from 15 to 66 pg/ml using the same assay conditions but required 500 or 1000 µl of plasma. Unlike adult females in which there is a pronounced seasonal cycle in plasma estradiol [23], there was no evidence for seasonal variation in plasma estradiol in immature female alligators (Table 1).

Tissue Expression of Aromatase

The quantitative kinetic reverse transcription PCR analysis of aromatase mRNA isolated from testes, ovaries, brain, oviduct, kidneys, and liver are presented in Table 2 and Figure 3. The Ct values presented in Table 2 represent the mean cycle number at which each sample's fluorescent signal is greater than the experimental noise threshold for the assay. For mRNA transcripts of 1–2 kilobases, a Ct in the range of 28–32 represents a copy number of approximately 4800 molecules. Thus, for the 20 ng input of DNA, there is a nearly 600-fold higher aromatase mRNA level in ovaries relative to testes and nearly a 10-fold higher expression in the brain than in the testes (Table 2 and Fig. 3). Low levels of aromatase transcript were found in kidneys, with no detectable levels found in liver at this input level. The amplification efficiencies of the PCR products were identical, as indicated by serial dilution of input cDNA from each tissue.


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  		TABLE 2. Levels of aromatase mRNA transcript in alligator tissues



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  		FIG. 3. Transcript abundance for the CYP19 gene expressed in tissues from six mature alligators (three males and three females). Transcript abundance is expressed relative to the transcript abundance of ß-actin in each tissue ({Delta}Ct; see Table 2) and then normalized to the expression level in the kidney ({Delta}{Delta}Ct; Table 2). No expression was found in the liver. The SEM is included with each mean but is too small to be seen on this logarithmic scale, except for the oviduct values

Tissue Activity of Aromatase

Although tritiated water release was detected in incubations conducted with alligator testicular microsomes, giving calculated enzyme activities of 0.92–2.38 pmol mg-1 h-1, this essentially represented background in the assay and was not inhibited at any concentration of Fadrazole. In contrast, aromatase activity in ovarian follicular microsomes was 36.2 ± 1.6 pmol mg-1 h-1 and was inhibited by Fadrazole in a clear concentration-dependent fashion, with an apparent IC50 value of 25–30 nM (Fig. 4). Positive controls included human placental microsomes, which exhibited 100-fold higher aromatase activities (1780 ± 10 pmol mg-1 h-1) with a similar sensitivity to Fadrazole (IC50 ~ 10 nM; data not shown).



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  		FIG. 4. Plot of inhibition curves of ovarian and testicular tissue aromatase activity in the presence of increasing concentrations of the aromatase inhibitor (Fadrazole). The individual animals from which samples were collected are indicated by the different symbols. The number next to the symbols for the five male samples indicates total length of the alligators in inches


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Male alligators do have circulating estradiol but at such low concentrations it is unlikely to be detected by most assay systems. We had to go to extraordinary lengths to actually prove its presence in male alligator plasma. The discrepancy between our results (0.29–3.14 pg/ml) in male alligators (Table 1) and those of Guillette et al. [28–32] (10–60 pg/ml) is difficult to explain. The difference could be due to the different assay systems used or to the possible endocrine disrupting effects of environmental contaminants in some alligator populations in Florida wetlands, as suggested by the authors [28–32]. The Florida group used a conventional estradiol RIA and extracted only 100–200 µl plasma. Without careful controls, spuriously high values could be obtained using this assay system. Nevertheless, male alligators in south Louisiana have insubstantial amounts of estradiol in the blood. The results do not support the contention that these very low concentrations of circulating estradiol in adult male alligators are derived from testicular secretion but suggest rather that circulating estradiol in male alligators is of extragonadal origin. The ratio of testosterone to estradiol in the circulation is enormous. In one large male, plasma testosterone was over 100 ng/ml but estradiol was only 0.95 pg/ml, a 100 000-fold difference. If there is extragonadal aromatization of this testosterone, it is not very active. These results do not rule out however a developmental change in circulating estradiol, as seen in the zebra finch [16]. The juvenile male alligators did have significantly higher estradiol levels than adults collected at the same time of year, suggesting that like birds there is a developmental change. We have attempted without success to measure estradiol in neonate male alligators (~20 cm total length) using this ultrasensitive assay [27]. The smallest male alligator in our study was 53 cm total length, and it is possible that alligators in the 25–45 cm range do have elevated estradiol.

Aromatase activity in alligator ovarian tissue was remarkably consistent among the three samples tested, and all three preparations showed similar inhibition curves in the presence of Fadrazole. This inhibition of alligator ovarian aromatase by Fadrazole is virtually identical to that observed in the turtle Malaclemys terrapin [38] and close to that seen in mammalian ovarian and placental preparations [39]. Apparent enzyme activity in the five testicular preparations was not significantly different from background and was not affected by the inhibitor. Thus, the testes of the alligator appear to lack significant aromatase activity and are unlikely to contribute to circulating levels of estradiol, which were in the low picogram per milliliter range. Similar conclusions have been reached with respect to avian testes [14].

It remains to be proven that estradiol is essential for spermatogenesis in nonmammalian vertebrates, but an argument for a role in male lizards has been proposed [12, 22]. If the alligator testis (and possibly bird testis) is not producing significant levels of estradiol, a number of interesting questions can be asked. Is spermatogenesis in alligators (or birds) estradiol dependent? If spermatogenesis is estradiol dependent, does it rely on an extragonadal source of the steroid? Tsai et al. [40] demonstrated in the turtle that the pituitary is exquisitely sensitive to estradiol but does not respond to testosterone. Testosterone has to be converted to estradiol by pituitary aromatase to inhibit LH secretion. If the alligator pituitary shows a similar mechanism of gonadotropin regulation, this might help explain the circulating estradiol in males. The seasonal variation in estradiol concentrations in both adult and immature male alligators is difficult to explain but may be related to the reproductive activity and presumably increase in extragonadal synthesis that occurs from April though May [23].

Extragonadal sources of aromatase activity have been well recognized in mammals [41], birds [16], reptiles [38], and fish [42], particularly in brain. Aromatase is also expressed in alligator embryonic brain but not in adult fat tissue, adrenal gland, liver, or kidney [35] (this study). Embryonic alligator brain tissue of both sexes does have measurable aromatase activity [43], but activity or expression of aromatase has not been studied in juvenile or adult alligator brain or pituitary. Alligator embryonic mesonephros-gonad tissue free of adrenal tissue shows a distinct sex difference in aromatase expression. Tissue from female alligator embryos exhibits gradually increasing aromatase mRNA expression during ovarian differentiation, whereas aromatase expression in tissue from male embryos remains at baseline [35]. Milnes et al. [43] reported a similar sex bias when measuring aromatase activity in alligator gonadal-adrenal-mesonephric (GAM) tissue. Embryonic female GAM tissue exhibited increasing activity with stage of development, whereas tissues from male embryos remained unchanged throughout development.

The current data indicate the sensitivity of alligator aromatase to inhibition by the commonly used potent aromatase inhibitor Fadrazole. These observations are important for at least two reasons. First, ambiguity exists concerning the role of embryonic ovarian aromatase expression on the sexual differentiation of crocodilians. The most convincing evidence for the functional importance of endogenous estrogen synthesis in the differentiation of the reptilian ovary comes from studies in turtles. Several laboratories have induced sex reversal among offspring of turtles from eggs at female-inducing incubation temperatures by application of aromatase inhibitors at critical developmental stages [44, 45]. In contrast, sex reversal of alligator embryos using aromatase inhibitors has been universally unsuccessful. Lance and Bogart [46] used a variety of inhibitors, including Fadrazole, at several different concentrations but were able to demonstrate only a disruption of ovarian development, even at the highest levels used. The data presented here suggest that Fadrazole is at least an effective inhibitor of alligator aromatase within the range of potency of mammalian [39] and more importantly turtle [38] aromatases. Thus, the lack of effective sex reversal in alligator embryos treated with Fadrazole at least is unlikely to represent a failure to inhibit aromatase activity but may be due to a dose effect. Successful sex reversal of female chicken embryos required a 1-mg dose [47], whereas the highest dose tested in alligator embryos was only 200 µg [46]. Second, tritiated water can be released as a result of nonspecific metabolism of [1ß-3H]-androstenedione by tissues such as embryonic liver, most of which cannot be suppressed by Fadrazole (unpublished results). Similarly, the very low levels of aromatase activity in alligator testicular tissue were unaffected by Fadrazole. Therefore, testis is unlikely to be a source of true aromatase activity or estrogen synthesis. Liver from adult alligator showed no detectable aromatase mRNA expression, supporting this interpretation. The lack of an effect of the highest dose of Fadrazole on the already very low aromatase activity in the alligator testis preparations therefore suggests that the tissue lacks a true aromatase. Unfortunately, few studies that have been conducted using this methodology with reptilian tissues have employed this important control.

The results from the aromatase mRNA expression studies again emphasize the enormous difference between ovary and testis and corroborate aromatase activity measures. Although testicular tissue had higher expression of aromatase mRNA than kidney tissue, expression in testicular tissue was 600-fold lower than that in ovarian tissue and 10-fold lower than that in brain (Table 2 and Fig. 3). Given that the detection limit of aromatase mRNA for a 20-ng cDNA input is about 40 molecules, it seems unlikely that these levels of expression are sufficient to produce the amount of circulating estradiol found in male alligators. Thus, alligator testicular tissue has little aromatase activity and little aromatase mRNA expression and probably does not secrete estradiol to any significant degree. The role of alligator brain and pituitary aromatase in estradiol secretion still needs to be addressed.


   ACKNOWLEDGMENTS
 
We thank Dr. Ruth Elsey, Phillip (Scooter) Trosclair III, and the staff of the Rockefeller Wildlife Refuge for their help in all aspects of alligator sampling and Carol Rutherford for help with the testosterone assays.


   FOOTNOTES
 
1 This research was supported in part by an NSF grant (IBN96-04265) to A.R.P. This is contribution 04-595 from the Center of Marine Biotechnology, Maryland Biotechnology Institute, University of Maryland. Back

2 Correspondence: Valentine A. Lance, Center for Reproduction of Endangered Species, P.O. Box 120551, San Diego, California 92112. FAX: 619 557 3959; lvalenti{at}sunstroke.sdsu.edu Back

Received: 20 February 2003.

First decision: 18 March 2003.

Accepted: 22 May 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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