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Research Article |
Department of Animal Science, 3
Department of Population Health & Reproduction,4 School of Veterinary Medicine, University of California, Davis, California 95616
ABSTRACT
Boars have high concentrations of plasma and testicular estrogens, but how this hormone is involved in feedback regulation of the gonadotropins and local regulation of testicular hormone production is unclear. The present study examined the effects of reducing endogenous estrogens by aromatase inhibition on concentrations of plasma LH and FSH and on testicular and plasma concentrations of testosterone (T) and immunoreactive inhibin (INH). Thirty-six littermate pairs of boars were used. One boar from each pair was assigned to the control group (vehicle); the other boar to the treatment group (aromatase enzyme inhibitor, Letrozole, 0.1 mg/kg body weight [BW]). Weekly oral treatment started at 1 wk of age and continued until castration at 2, 3, 4, 5, 6, 7, or 8 mo. Plasma concentrations of gonadotropins, INH, T, estradiol (E2), and estrogen conjugates (ECs) were determined. Testicular tissue was collected at castration for determination of INH and T and for confirmation of reduced aromatase activity. The acute effects of aromatase inhibition on gonadotropins were monitored in two adult boars treated once with Letrozole (0.1 mg/kg BW). Treatment with the aromatase inhibitor reduced testicular aromatase activity by 90% and decreased E2 and ECs without changing acute, long-term, or postcastration LH and FSH. Plasma T, testicular T, and circulating INH concentrations did not change. Testicular INH was elevated in treated boars compared with controls. In conclusion, estrogen does not appear to play a regulatory role on gonadotropin secretion in the developing boar. This is in direct contrast to findings in males of several other species.
estradiol, follicle-stimulating hormone, inhibin, luteinizing hormone, testosterone
INTRODUCTION
Estrogen is increasingly recognized as an important hormonal regulator of male reproductive function, as highlighted through studies with mouse knockout models. Estrogen-receptor knockout mice that are deficient in estrogen receptor alpha (ESR1) develop testicular atrophy with tubular degeneration and reduced sperm production and fertility [1, 2]. Researchers have demonstrated that this results from a lack of fluid reabsorption in the efferent ductules [3]. In contrast, estrogen receptor beta (ESR2) knockouts are phenotypically normal [4], although the immunolocalization of the ESR2 in Sertoli cells, Leydig cells, and developing germ cells of rat testes is consistent with a role of estrogen and ESR2 in testicular function. Aromatase (CYP19A1) is the enzyme solely responsible for the bioconversion of androgens to estrogens [5]. The CYP19A1 knockout mice, the phenotype of which is caused by targeted disruption of the CYP19A1 gene, are incapable of synthesizing estrogens. Initially, these mice are fertile, but testis weight and spermatogenesis are compromised with advancing age [6]. In CYP19A1 knockout mice, estradiol (E2) levels are undetectable, whereas levels of testosterone (T), LH, and FSH are elevated [7].
The role of estrogen in the regulation of gonadotropins has been investigated in several species [8–11] and is believed to influence the secretion of LH and/or FSH. In adult male mice [12, 13], monkeys [13], and men [8], E2 has a suppressant effect, whereas in stallions [10, 11], it has a stimulatory effect on LH and/or FSH secretion. Gonadotropins are essential for testicular growth and development and for the support of testicular factors necessary for the initiation and maintenance of spermatogenesis [14]. An understanding of the underlying factors controlling gonadotropin secretion therefore is important to understanding male reproductive development.
The importance of estrogen in boars is especially intriguing, because these animals have higher levels of estrogens than are found in males of most species. In fact, plasma estrogen levels are well above those in sows during estrus [15]. Numerous studies have assessed and profiled plasma reproductive hormones in the developing boar [16–21] and although all have confirmed relatively high estrogen levels, none have attempted to explore the role of estrogen as a regulator of hormone dynamics.
The present study elucidates the importance of estrogen in the regulation of gonadotropin secretion and in the maintenance and regulation of T and inhibin concentrations in the developing boar. To our knowledge, these are the first experiments in pigs that use a potent and specific aromatase enzyme inhibitor (Letrozole, CGS 20 267; Ciba-Geigy) to evaluate the chronic reduction of estrogen on the hypothalamic-pituitary axis of the boar.
MATERIALS AND METHODS
Animals
Thirty-six littermate pairs of boars were used in the present study. The boars were born and raised at the University of California, Davis, Swine Facility and were from established lines developed from Durocs, Hampshires, Yorkshires, and Pietrains provided by PIC USA (a division of Sygen International). Experiments were conducted in accordance with the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching [22] and approved by the Animal Use and Care Advisory Committee at the University of California, Davis. One animal from each pair was assigned to the control group (dosed orally with corn oil). The other animal was assigned to the treatment group; these boars were treated orally with a nonsteroidal aromatase inhibitor, Letrozole [CGS 20 267; 4–4'-(1H-1,2,4-triazol-1-yl-methylene)-bis-benzonitrile; Ciba-Geigy]. Treatment was given at 0.1 mg/kg body weight (BW) starting at 1 wk of age and continued weekly until 2, 3, 4, 5, 6, 7, or 8 mo of age, at which time boars were castrated. The frequency and dose of 0.1 mg/kg BW were chosen based on preliminary studies. Four littermate pairs were castrated at each time point except 2 mo of age, when three boars from each of four litters were assigned to the control and the corresponding littermates to the treatment. Hence, eight boars were castrated at each age except 2 mo, when 24 boars were assigned for a total of 72 boars, or 36 pairs.
Testicular tissue from each boar was stored in cryovials, snap-frozen in liquid nitrogen, and stored at –80°C before homogenization and determination of aromatase activity and testicular hormone content. Blood was collected by jugular venipuncture in heparinized, 10-ml vacutainer tubes at the same time of day on Day –2, Day –1, and on the day of castration (Day 0), as well as on Days 7, 8 and 9 postcastration. Blood was spun at 1300 x g and 4°C in a Sorvall RT6000B centrifuge (Du Pont) for 20 min to separate plasma. Plasma from precastration samples was frozen at –20°C until assayed for LH, FSH, immunoreactive inhibin (INH), T, E2, and estrogen conjugates (ECs) using RIA. Postcastration samples were assayed for LH and FSH. Animals were classified as prepubertal (2–3 mo), pubertal (4–5 mo), and postpubertal (6–8 mo) based on sperm production and behavior observed in contemporary herdmates.
In a short-term study to examine the acute effects of aromatase inhibition, two boars (ages, 12 and 14 mo) were treated with a one-time oral dose of Letrozole (0.1 mg/kg BW), and blood was collected from the ear vein or by jugular venipuncture at –24, –0.5, 0, 2, 6, 24, 48, and 168 h relative to treatment. Recovered plasma was assayed for LH and FSH to study the acute effects of estrogen reduction on the gonadotropins and for E2 to confirm a decrease following treatment with the aromatase inhibitor.
Testicular Homogenization
Tissue was homogenized in 2 ml of 0.1 M phosphate buffer (pH 7.0) containing 0.01% thimerosal (Sigma) using a Barnant mixer-motor and pestle (General Electric) for approximately 2–3 min or until tissue was completely ground. The suspension was then spun at 13000 x g in a Sorvall RC-5B centrifuge (Du Pont) for 20 min, and supernatant was collected and frozen at –20°C until assayed for E2, T, ECs, INH, and protein concentration.
Protein Assay
Homogenized testicular samples were diluted 1:100 in 0.1 M phosphate buffer containing 0.01% thimerosal before protein determination using the Bradford Protein Assay method (Bio-Rad Laboratories, Inc.). Protein values were used to normalize hormone (E2, T, ECs, and INH) concentrations in testicular samples.
Radioimmunoassay
All assays were validated. Parallelism of boar plasma with assay standards was confirmed for all hormone assays. Parallelism also was confirmed for testicular homogenate supernatant for the applicable hormone assays (INH, T, E2, and ECs). Recoveries of spiked standards from plasma or testicular extracts were greater than 90% for all hormones assayed.
LH Plasma LH concentrations were measured in duplicate in plasma samples by RIA using a mouse monoclonal anti-bovine LH (518B7, Roser [23]) and porcine LH (EX275A, Papkoff) standards. Porcine LH was iodinated by the iodogen method as described previously [23]. Standards were made up in RIA buffer (10 mM sodium phosphate buffer with 0.5% BSA, 2 mM EDTA, and 0.1% thimerosal; pH 7.5). One-hundred microliters of sample or standard were added to 100 µl of RIA buffer and 100 µl of antibody (1:350000). Fifty microliters of iodinated porcine LH was added at 20000 counts/tube. Tubes were incubated overnight at room temperature (RT). One-hundred microliters of a 1.0% normal mouse serum (Antibodies, Inc.) and 1 ml of a 1:300 dilution of goat anti-mouse antibody (Antibodies, Inc.) in 0.05 M phosphate buffer and 5.0% polyethylene glycol (Research Products International Corp.) were added to each sample on Day 2 of the assay. Samples were incubated for 2 h at RT followed by centrifugation in a Sorvall RC-3B centrifuge (Du Pont) at 1160 x g for 20 min at 4°C. Pellets were counted on a Micromedic gamma counter. The sensitivity of the assay was 0.2 ng/ml, and the intra- and interassay coefficients of variation were 2.9% (n = 8) and 5.8% (n = 3), respectively.
FSH Porcine FSH was measured in duplicate using 100-µl samples of plasma by RIA with a rabbit anti-porcine FSH (R285; courtesy of H. Papkoff, University of California, Davis, CA) and iodinated porcine FSH (EX274B, Papkoff) standards. One-hundred microliters of RIA buffer and 100 µl of porcine FSH antibody (1:1500) were added, and samples were incubated overnight at RT. On the second day, 100 µl of porcine FSH trace was added at 20000 counts/tube, and samples were incubated for 3 days at 4°C. On the fifth day, 100 µl of a 1% normal rabbit serum (Sigma) and 1 ml of goat anti-rabbit antibody (1:300 dilution; Antibodies, Inc.) were added to each tube. Samples were incubated for 3–4 h at RT and centrifuged for 20 min at 1160 x g. Pellets were counted on a Micromedic gamma counter. All samples were analyzed in a single assay. The sensitivity of the assay was 0.4 ng/ml, and the intra-assay coefficient of variation was 2.9% (n = 6).
Immunoreactive inhibin Inhibin was measured in duplicate using 100-µl samples of plasma or testicular homogenate supernatant by RIA as described previously [24]. The sensitivity of the assay was 0.15 ng/ml, and the intra- and interassay coefficients of variation were 2.4% (n = 6) and 4.9% (n = 3), respectively, for plasma samples. The intra- and interassay coefficients of variation for the testicular homogenates were 2.9% (n = 6) and 5.8% (n = 2), respectively.
Testosterone
Testosterone was measured in duplicate using 50-µl samples of plasma or testicular homogenate supernatant by RIA using sheep anti-testosterone-11-BSA (Niswender #S250; courtesy of G. Niswender, Colorado State University, Fort Collins, CO [25]) and T trace (testosterone 1,2,6,7-3H, NET370; Perkin-Elmer) as described previously [26]. Samples were diluted in a 0.01 M phosphate buffer with 0.1% gelatin and 0.01% thimerosal (pH 7.0) when necessary to fall within assay parameter limits. Cross-reactivities for the antibody were determined previously to be 100% for T; 69% for dihydrotestosterone; 14% and 20% for 3
- and 3ß-androstanediol, respectively [25]; and 8.6% for nortestosterone as determined in our lab. The sensitivity of the assay was 0.1 ng/ml, and the plasma intra- and interassay coefficients of variation were 4.3% (n = 6) and 9.2% (n = 10), respectively. Extraction efficiencies were 85% and 100% for plasma and testicular samples, respectively. All testicular homogenates were analyzed in a single assay, and the intra-assay coefficient of variation was 2.6% (n = 6).
Estradiol Estradiol was measured in duplicate using 200-µl samples of plasma or testicular homogenate supernatant by RIA using a sheep anti-estradiol 17ß-6-BSA (Niswender #244; courtesy of G. Niswender [27]) and tritiated E2 (estradiol 1,2,6,7-3H, NET317; Perkin-Elmer) as described previously [26]. Plasma sample volumes were adjusted when necessary to fall within assay parameter limits. The antibody has a 3% cross-reactivity with estrone [28]. The sensitivity of the assay was 10–12 pg/ml, and the intra- and interassay coefficients of variation were 4.4% (n = 6) and 8.6% (n = 10), respectively, for plasma samples. Extraction efficiencies for plasma and testicular samples were approximately 78% and 100%, respectively. Testicular homogenate samples were analyzed in a single assay, and the intra-assay coefficient of variation was 1.2% (n = 6).
Estrogen conjugates Estrogen conjugates were measured in duplicate using 50-µl samples of plasma or testicular homogenate supernatant by RIA using rabbit anti-estrone-3-glucuronide (Munro R-583, 1:12000 dilution; courtesy of C.J. Munro, Clinical Endocrinology Lab, University of California, Davis, CA) and tritiated estrone sulfate (estrone sulfate 1,2,6,7-3H, NET203; Perkin-Elmer) as described previously [26, 29]. Samples were diluted in 0.1 M Tris with 0.1% gelatin (pH 8.4) when necessary to fall within assay parameter limits. The sensitivity of the assay was 0.3 ng/ml, and the intra- and interassay coefficients of variation were 6.6% (n = 6) and 13.1% (n = 11), respectively, for the plasma samples. The testicular homogenate samples were analyzed in a single assay, and the intra-assay coefficient of variation was 3.5% (n = 4).
Microsome Preparation
Snap-frozen testicular tissue was homogenized on ice in 0.1 M potassium phosphate buffer (pH 7.4) containing 20% glycerol (Sigma), 5 mM ß-mercaptoethanol (Sigma), and 0.5 mM phenylmethylsulfonyl fluoride (Sigma) as described previously [30, 31]. Tissue samples were sonicated briefly, and debris and mitochondria were removed by centrifuging at 15000 x g in an Avanti J-25I centrifuge (Beckman Instruments, Inc.) for 10 min. Supernatant was centrifuged at 100000 x g in a Beckman L8-70M ultracentrifuge (Beckman Instruments, Inc.) for 60 min, and the pellet was resuspended in buffer. Protein concentration was determined for each sample using the Bicinchoninic Acid Protein Assay Reagent (Pierce). Samples were aliquoted and stored at –80°C until assayed for aromatase activity. The purity of the microsomal fractions has been demonstrated previously [32].
Aromatase Activity
Aromatase activity was determined in the microsomal fraction of testicular tissue by the tritiated water assay using [1ß-3H]androstenedione (24.7 Ci/mmol; New England Nuclear) as described previously [30, 31, 33]. Briefly, 100 µg of microsomal protein were incubated for 1 h at 37°C in the presence of 300 nM androstenedione (3.3 x 1012 dpm; New England Nuclear) and a generating system consisting of 17 mM glucose-6-phosphate, 1 mM NADPH, 2 mM NADP, and 1 U of glucose-6-phosphate dehydrogenase (Sigma). Some samples also were incubated with Letrozole in addition to the generating system to confirm in vitro inhibition of the enzyme activity being measured. The reaction was stopped with 0.25 ml of 30% trichloroacetic acid and extracted with 1 ml of chloroform (Fisher). Charcoal (5%; Fisher) and dextran (0.5%; Sigma) were added to the aqueous phase, and the samples were then spun at 2000 x g for 30 min. Five-hundred microliters of supernatant were then added to 10 ml of scintillation cocktail (EcoLite scintillation cocktail; ICN) and counted in a Tri-Carb 2000 Liquid Scintillation Analyzer (United Technologies Packard).
Western Blot
Microsomal proteins (10 µg) were separated using 8% SDS-PAGE as described previously [31]. After separation, proteins were transferred to polyvinylidene fluoride membranes (Immobilon P; Millipore Corp.). Membranes were blocked in 20% dried milk, then incubated with polyclonal antisera raised against recombinant human CYP19A1 (1:3000; courtesy of Dr. N. Harada, Fujita Health University, Japan) at RT in PBS with 0.1% Tween 20 (Amresco). The CYP19A1 was visualized using a donkey anti-rabbit horseradish peroxidase-linked IgG whole antibody (Amersham) at 1:10000 dilution. Membranes were washed, and the target protein signal was generated by chemiluminescence using luminol reagent (New England Nuclear). The immunodetectable bands were quantified by densitometry of the film. Blots were reprobed for cytochrome b5, which also is expressed at high levels in boar testes, and were quantified by densitometry. No differences were found in cytochrome b5 levels with either treatment or age (data not shown).
Data Analysis
Plasma hormone values for each boar were averaged for Days –2, –1, and 0 precastration and for Days 7, 8, and 9 postcastration before statistical analysis. Data were analyzed by a two-way ANOVA with age and treatment as main effects using the general linear model procedure (SAS Statistical Software; SAS Institute, Inc.). When appropriate, pairwise comparisons were made for control and treated littermates using least-squares means. Results are expressed as the least-squares means ± pooled SEM. Data from the short-term study for determination of acute effects on gonadotropins were analyzed using a one-way ANOVA, and gonadotropin concentrations posttreatment were compared with time 0 (just before treatment) using the Tukey studentized range test.
RESULTS
Plasma and Testicular Hormone Concentrations
Gonadotropins
Aromatase inhibition did not affect short-term or long-term circulating gonadotropin concentrations (Fig. 1, A–C). Gonadotropin concentrations were, however, influenced by age (P = 0.0004 and P < 0.0001 for LH and FSH, respectively) (Fig. 1, A and B). Although LH was highest just before puberty (3 mo; P 
0.03), this trend was more pronounced in Letrozole-treated boars (Fig. 1A). Generally, FSH was lower before puberty but increased approximately 40% from 2 to 5 mo of age (P < 0.0001) (Fig. 1B). A significant age by treatment interaction was found for FSH because of the significant difference in plasma FSH concentrations at one age point (8 mo) (Fig. 1B). Castration increased both LH and FSH, as expected (Fig. 1, A and B). Postcastration gonadotropin concentrations in control and aromatase-inhibited boars were not different. Acute effects of aromatase inhibition on plasma gonadotropin concentrations were not observed (Fig. 1C), although E2 was decreased by approximately 90%, declining dramatically within the first 24 h of treatment from 465.39 ± 116.03 to 36.71 ± 11.48 pg/ml and then remaining at this level up to 168 h posttreatment.
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Immunoreactive inhibin Aromatase inhibition did not significantly alter plasma INH concentrations (Fig. 2A) but caused an overall 55% increase in testicular INH concentrations (P = 0.046) (Fig. 2B). Both plasma and testicular concentrations of INH initially were high but declined rapidly with age (P < 0.0001 and P = 0.033, respectively).
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Testosterone
Aromatase inhibition did not affect plasma or testicular T concentrations (Fig. 3, A and B), although both were affected by age of the boar. Plasma T concentrations increased by 25-fold (P < 0.0001) (Fig. 3A), whereas testicular T concentrations increased by twofold from prepuberty to puberty (P = 0.004) (Fig. 3B). Both significantly decreased thereafter (P 0.0004).
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Estradiol
Aromatase inhibition significantly reduced plasma E2 concentration during puberty and early into postpuberty in these boars (P 0.05) (Fig. 4A). Furthermore, in treated boars, no difference was found in E2 concentration with development (age), because E2 remained low during the entire study. In control boars, plasma E2 concentrations initially were low (2 and 3 mo, 13.8 ± 2.6 and 10.5 ± 4.5 pg/ml, respectively), increased at puberty (4 and 5 mo, 17.1 ± 4.5 and 44.4 ± 4.5 pg/ml, respectively; P 0.02), and decreased again during the postpubertal period (8 mo, 8.4 ± 4.5 pg/ml; P 0.01) (Fig. 4A), as was the case with plasma and testicular T concentrations (Fig. 3). The decrease in E2 concentrations from 5 to 8 mo in control boars was consistent with the reduction in testicular aromatase activity at this time period (Fig. 5A).
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The testicular E2 concentrations in control and aromatase-inhibited boars (Fig. 4B) were consistent with those of plasma E2 (Fig. 4A). Estradiol concentrations were suppressed dramatically by Letrozole and did not differ during the entire duration of the present study in aromatase-inhibited boars. In these boars, testicular E2 concentrations were reduced to 12% of the concentration in control littermates at the time of puberty. In the control boars, testicular E2 concentrations were highest at puberty (804.7 ± 11.08 pg/mg protein at 5 mo) (Fig. 4B) but dropped significantly by 6–8 mo of age. This was consistent with aromatase activity (Fig. 5A).
Estrogen conjugates Concentrations of plasma ECs were reduced in Letrozole-treated boars compared with control littermates during most of the developmental period (Fig. 4C). As with plasma E2, EC concentrations in plasma of aromatase-inhibited boars did not vary with age. In the control group of boars, plasma EC concentrations initially decreased from 2 to 3 mo, peaked at 5 mo (6.5 ± 0.5 ng/ml, P < 0.0001), and declined thereafter. This pattern was similar to that of testicular E2 concentration (Fig. 4B).
As in the case of plasma ECs, Letrozole treatment decreased testicular EC concentrations before and at the onset of puberty (P 0.05) (Fig. 4D). In aromatase-inhibited boars, testicular EC concentration remained low and did not change with age. In the control boars, testicular EC concentration declined by 90% from 2 mo (0.3 ± 0.03 ng/mg protein) to 8 mo (0.04 ± 0.03 ng/mg protein) of age (P = 0.0002) (Fig. 4D). The decline in testicular EC concentration closely paralleled the decline in aromatase activity (Fig. 5A).
Aromatase Activity and Protein Levels
Treatment with the aromatase enzyme-inhibitor Letrozole significantly reduced aromatase activity in testicular tissue of boars overall by 83% (Fig. 5A). A significant effect of the age by treatment interaction was detected for aromatase activity (P = 0.004). Testes from Letrozole-treated boars before and at the onset of puberty (
4–5 mo) had significantly reduced aromatase enzyme activity compared with that of control littermates (P 0.05). This reduction was 92% at 2 mo (168.1 ± 16.5 pmol (mg protein)–1 h–1 in control boars vs. 13.7 ± 16.5 pmol (mg protein)–1 h–1 in Letrozole-treated littermates) and 80% at 5 mo of age (79.0 ± 16.5 pmol (mg protein)–1 h–1 in control boars vs. 15.5 ± 16.5 pmol (mg protein)–1 h–1 in treated littermates). Furthermore, an overall 90% decrease in aromatase activity was found from 2 to 8 mo of age (P = 0.0004).
In contrast to the reduction in aromatase activity, aromatase protein levels were actually increased by Letrozole treatment as determined by Western blot analysis (Fig. 5B) and confirmed by densitometry (P < 0.025).
DISCUSSION
To our knowledge, this is the first study to investigate the role of endogenous estrogen in the regulation of both LH and FSH secretion in the boar. Surprisingly, neither chronic nor acute inhibition of estrogen synthesis altered LH or FSH secretion. The postcastration rise in the gonadotropins was similar between control and treated groups, again indicating that removal of estrogen did not affect gonadotropins. Previous research [34, 35] in gonadectomized, estradiol benzoate-treated boars has shown no change in LH levels, but effects on FSH were not determined. Estrogen response elements were not found on porcine LH and FSH genes [36, 37]. Hence, the boar is unusual among male mammals studied thus far in which estrogen plays a role in the regulatory feedback effect at the level of the hypothalamus and/or pituitary. Aromatase inhibition that similarly suppressed endogenous estrogens, increased both serum LH and FSH concentrations in men [8] and LH concentrations in early and midpubertal boys [9]. The same result was demonstrated by use of an aromatase inhibitor in adult dogs [38]. This supports the theory that in both the dog and the human, estrogen is acting directly at the hypothalamus and/or pituitary to suppress gonadotropin secretion. This also appears to be the case for mice, in which E2 suppresses FSH mRNA [12], and for the castrated ram [39] and male rhesus macaque [13], in which exogenous E2 has a negative effect on LH secretion. In contrast, estrogen appears to have a positive-feedback effect on gonadotropins in adult stallions, as exemplified by reduced LH concentrations following aromatase enzyme inhibition [10] and by an increase in both LH and FSH concentrations in geldings treated with exogenous E2 [40]. Furthermore, E2 incubated with stallion pituitary cells increased GnRH-induced LH secretion [11]. Therefore, whereas it appears that significant species differences exist in the feedback effects of E2 at the hypothalamus-pituitary axis, it is interesting to note that no feedback effect was demonstrable in the developing boar in the present study. To our knowledge, this is the only male of any domestic species in which estrogen does not appear to influence gonadotropin secretion during development.
In contrast to the differences in the role of estrogen as a feedback regulator, the boar resembles other males in the inverse relation between inhibin and FSH and in the regulatory effect of inhibin on FSH secretion. In the present study, it is apparent that in the boar, INH and FSH are inversely related, with INH concentrations highest before the onset of puberty and declining thereafter, whereas FSH concentrations display the exact opposite trend, increasing at the onset of puberty. This trend is consistent with the postcastration rise in FSH and also is seen in plasma INH concentrations in 8-mo-old boars, although the difference is not dramatic. Lack of a significant difference potentially may result from the fact that the inhibin assay used herein measures INH rather than individual concentrations of inhibin A or B, the bioactive inhibins. The gonadotropins (especially FSH) are major regulators of inhibin subunit gene expression, and transcriptional regulation and control of repressor molecules can alter levels of INH (for review, see [41]). Moreover, synthesis and secretion of inhibins A and B are regulated differentially, but the mechanisms of regulation are not yet understood (for review, see [41]).
In addition to the endocrine feedback regulation effects of FSH on inhibin, and vice versa, local testicular regulation of inhibin production may, in part, be under the control of E2. In the present study, testicular INH concentrations in aromatase-inhibited boars were higher than those in control littermates. This difference could not be attributed to differences in plasma FSH concentrations between the two groups, but it could result from the decrease in local action of E2. Plasma INH concentrations were high before the onset of puberty, coinciding with relatively lower plasma E2 concentrations. This inverse relationship between inhibin and E2 is substantiated by studies of aromatase inhibition in developing boys that showed an increased concentration of inhibin B that was not accompanied by decreased FSH [9]. In contrast, previous research in the rat demonstrated a positive effect of E2 on local inhibin production by Sertoli cells [42] and on subunit mRNA levels by granulosa cells [42, 43] in vitro. The same effect was seen in stallions in which treatment with an aromatase enzyme inhibitor decreased plasma inhibin concentrations without changing FSH concentrations [10]. Hence, evidence from the current and previous studies support a possible role for E2 in the regulation of local inhibin production.
Along with inhibin, T is another likely regulator of gonadotropin secretion in the boar. In the present study, the general lack of change in plasma LH and FSH concentrations after inhibition of estrogen synthesis was consistent with the lack of change in plasma T concentration. It is possible that T regulates gonadotropin output by the gonadotropin cells in the pituitary or affects GnRH release at the hypothalamus. The role of T as a feedback regulator of LH in the boar is supported by the rise in postcastration gonadotropin concentrations, by data showing marked suppression of both LH and FSH after immunization of prepubertal boars against T [44], and in studies of gonadectomized boars in which treatment with exogenous T reduced LH concentrations [34, 45]. Reduced estrogen synthesis did not significantly alter circulating and testicular T concentrations in the developing boar. In neonatal pig testes, activity of 17,20-lyase, the enzyme that regulates androgen synthesis, is 200-fold higher compared with aromatase enzyme activity [32]. By this estimate, estrogen synthesis represents less than 1% of total androgen synthesis in the postnatal period. Testicular tissue concentrations of E2 and ECs were substantially lower than T concentrations on average. Consistent with tissue concentrations observed in the present study, Christenson et al. [46] estimated that the rate of testicular production of E2 was less than 4% of the rate of testicular T production in peripubertal boars. Even though circulating levels of ECs and T were more similar, this likely reflects the very significant differences in half-lives of free versus conjugated steroids. Furthermore, T represents only a small fraction of the many androgens in boars [17]. Therefore, inhibition of androgen metabolism to estrogen would not be expected to have any marked impact on either testicular T or total testicular androgen levels in the boar, particularly because no increase was observed in LH trophic drive.
Testicular ECs in control boars followed the aromatase enzyme activity profile very closely. Therefore, as the testicular conversion of androgens to estrogens decreased with age, testicular EC concentrations also decreased. The fact that testicular EC concentrations did not follow plasma EC concentrations closely suggests that conjugation and deconjugation of estrogens may be occurring at different rates in the testes and other estrogen-conjugating organs, such as the liver or epididymis. Estrogens are conjugated in the periphery [47], within the Leydig cells [48], and by the epididymis, vas deferens, seminal vesicle, and prostate of boars [49]. Therefore, a majority of estrogens may be conjugated elsewhere.
It is interesting to note that despite the decrease in aromatase activity in testicular microsomes from Letrozole-treated boars, an increase in CYP19A1 immunoreactive protein was observed. The levels of other microsomal proteins, notably cytochrome b5, did not change, arguing against an inhibitory effect of this protein on activity as shown previously [50] and against error in loading gels as an explanation for the differences in CYP19A1 expression. The increase in CYP19A1 expression therefore might represent a local, positive effect of reduced E2 at the transcriptional level. Consistent with these data, treatment with Letrozole increased immunoreactive CYP19A1 and activity in brains of quail [51] and aromatase activity in guinea pig brain [52], in which the inhibitor was presumed to wash out of tissues during preparation, allowing activities to be measured [52]. Despite increasing the substrate concentration to 3 µM, activities in testis microsomes from Letrozole-treated boars remained lower than those from control boars (data not shown). At least some imidazole inhibitors bind porcine gonadal CYP19A1 with higher affinity than other aromatases [53], supporting the notion that Letrozole did not wash out during tissue processing. Whether the increase in CYP19A1 reflects transcriptional regulation in response to changes in the balance of sex steroids [54] or, perhaps, a decrease in protein turnover induced by Letrozole binding requires further study.
Aromatase inhibition was an effective tool by which to study the role of endogenous estrogens in male reproduction. Treatment with Letrozole, a nonsteroidal aromatase enzyme inhibitor, effectively reduced plasma estrogen levels in the adult stallion [10], adult monkey [8], and early and midpubertal boys [9]. In the present study, Letrozole significantly decreased testicular aromatase activity and dramatically reduced plasma and testicular concentrations of E2 and ECs in developing boars. This may have great implications for male reproduction, because E2 is important in various aspects of male reproduction, including sperm motility [55], germ cell survival [56], and testicular maturation [57].
The patterns and concentrations of hormones in the boars (especially for E2, estrone sulfate, and T) of the present study differ somewhat from previous patterns and concentrations reported by other researchers, although variability exists in these reports as well [16, 17, 21]. We attribute these differences to those in the breeds and lines of boars used in the present study compared with the others. Another factor, which may influence hormone concentrations, most notably T, is time of day [58]. Furthermore, the hormone concentrations, especially T and E2, will vary depending on when the boars reach puberty, and in turn, this is influenced by feed intake, housing (light vs. dark hours), genetics, and other management factors [59]. Data from the present study are consistent in terms of the rise in both E2 and T at puberty (4–5 mo); furthermore, samples were collected at the same time of day from all boars.
In summary, a dramatic decline in endogenous estrogen does not appear to have an effect on LH and FSH secretion in the developing boar, suggesting that estrogen is not a feedback regulator of gonadotropins in this animal during development. Other testicular hormones (T and inhibin) are more likely candidates for the regulation of gonadotropins in the boar. Hence, the developing boar is unique among males of domestic animals with regard to the feedback roles of testicular hormones on the hypothalamus-pituitary axis.
ACKNOWLEDGMENTS
We would like to thank Kent Parker, Animal Science Swine Facility Manager, and the personnel at the facility for the care and maintenance of the boars as well as for assistance in handling the animals for collection of blood and tissue samples. Thanks are extended to Chris Pearl, Megan McCarthy, Matt Rooney, and Judy Etchevveria for help with various parts of the present study. We would like to acknowledge the help of Dr. Tom Famula, Professor of Animal Science, for assistance with statistical analysis. Finally, we would like to thank Jo Corbin for running the Western blots and densitometry analysis, Lillian Sibley for technical assistance with the RIA procedures, and Samantha Mapes for help with microsomal preparations and the aromatase activity assays. Mention of commercial products in this article is solely for information purposes and does not constitute an endorsement by the authors.
FOOTNOTES
1 Supported by National Research Initiative Competitive grant 2002-35203-12606 from the USDA Cooperative State Research, Education, and Extension. 
2 Correspondence: Janet F. Roser, Department of Animal Science, University of California, One Shields Ave., Davis, CA 95616. FAX: 530 752 0175; jfroser{at}ucdavis.edu
106 DOI: 10.1095/biolreprod.105.043760
Received: 11 May 2005.
First decision: 17 June 2005.
Accepted: 9 September 2005.
REFERENCES
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E. E. At-Taras, T. Berger, M. J. McCarthy, A. J. Conley, B. J. Nitta-Oda, and J. F. Roser Reducing Estrogen Synthesis in Developing Boars Increases Testis Size and Total Sperm Production J Androl, July 1, 2006; 27(4): 552 - 559. [Abstract] [Full Text] [PDF]
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