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BOR - Papers in Press, published online ahead of print April 2, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.015578
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BIOLOGY OF REPRODUCTION 69, 390–397 (2003)
DOI: 10.1095/biolreprod.103.015578
© 2003 by the Society for the Study of Reproduction, Inc.

Ovary

Biochemical Assessment of Limits to Estrogen Synthesis in Porcine Follicles1

C.J. Corbin3, F.M. Moran3, J.D. Vidal3, J.J. Ford4, T. Wise4, S.M. Mapes3, V.C. Njar5, A.M. Brodie5, and A.J. Conley2,3

Department of Population Health & Reproduction,3 School of Veterinary Medicine, University of California, Davis, California 95616 Roman L. Hruska U.S. Meat Animal Research Center,4 ARS, USDA, Clay Center, Nebraska 68933 Department of Pharmacology & Therapeutics,5 University of Maryland School of Medicine, Baltimore, Maryland 21201


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Limits to estrogen production by early and late preovulatory porcine follicles were assessed by comparing enzymatic capacities for androgen (17,20-lyase) and estrogen (aromatase) synthesis in theca interna and granulosa, support of enzyme activities by the redox partner proteins NADPH-cytochrome P450 oxidoreductase (reductase) and cytochrome b5, and tissue-specific expression and regulation of these proteins. Parameters included follicular fluid (FF) estradiol and progesterone levels, theca and granulosa aromatase and reductase activities, and theca 17,20-lyase activity. Expression of proteins responsible for these activities, aromatase (P450arom) and 17{alpha}-hydroxylase/17,20-lyase (P450c17) cytochromes P450, reductase, and for the first time in ovarian tissues cytochrome b5, were examined by Western immunoblot and immunocytochemistry. Theca and granulosa aromatase activities were as much as 100-fold lower than theca 17,20-lyase activity, but aromatase was correlated with only the log of FF estradiol. Granulosa reductase activity was twice that of the theca, and cytochrome b5 expression was clearly identified in both the theca and granulosa layers, as was P450arom, but was not highly correlated with either 17,20-lyase or aromatase activities. Reductase expression did not change with stage of follicular development, but cytochrome b5, P450c17, and P450arom were markedly lower in post-LH tissues. These data indicate that aromatase and not 17,20-lyase must limit porcine follicular estradiol synthesis, but this limitation is not reflected acutely in FF steroid concentrations. Neither reductase nor cytochrome b5 appear to regulate P450 activities, but the expression of cytochrome b5 in granulosa and theca suggests possible alternative roles for this protein in follicular development or function.

estradiol, follicle, granulosa cells, ovary, theca cells


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Balanced androgen and estrogen synthesis is necessary for normal reproductive development [1–3] and function [4, 5]. Both biosynthetic reactions are catalyzed by cytochrome P450 enzymes; androgen synthesis by 17{alpha}-hydroxylase/17,20-lyase cytochrome P450 (P450c17; catalyzing both 17{alpha}-hydroxylating and 17,20-lyase activities) [6] and estrogen synthesis from androgens by aromatase cytochrome P450 (P450arom) [7]. The steroid-hydroxylating P450 enzymes are membrane bound; some are in the mitochondria, such as cholesterol side-chain cleavage, whereas others are found in the smooth endoplasmic reticulum (microsomal compartment). Both P450c17 and P450arom fall into this latter group [8–10]. Like all microsomal P450s, P450c17 and P450arom are not catalytically active unless associated in an enzyme complex with the accessory flavoprotein NADPH-cytochrome P450 reductase (reductase), a redox partner that shuttles electrons from NADPH to the P450 and subsequently catalyzes substrate oxidation [11]. Reductase is also an exceptionally conserved microsomal protein [12], and although there are other potential redox proteins, such as cytochrome b5 [6], the catalytic activity of microsomal P450s is entirely dependent on coupling to reductase. Thus, reductase availability could potentially limit 17,20-lyase and aromatase activities (i.e., androgen and estrogen synthesis) without affecting levels of P450c17 and P450arom enzyme protein levels. This limitation has not been found in the developing porcine testes [13], but to our knowledge the possibility has not been examined in ovarian follicular tissues. Similarly, no information exists on regulation or expression of either reductase or cytochrome b5 in follicular tissues, although cytochrome b5 can support 17,20-lyase activity [14, 15] and/or potentially inhibit the catalytic activity of P450arom [16], thereby potentially influencing sex steroid synthesis.

Despite species diversity in reproductive patterns, a preovulatory rise in follicular estrogen secretion is common among all mammals and is essential in coordinating many aspects of ovarian and reproductive function [5]. Thus, a critical aspect of ovarian physiology to be explored is which enzymatic step limits estrogen production by the preovulatory follicle: androgen synthesis (catalyzed by P450c17) or its metabolism to estrogen (catalyzed by P450arom). The major regulating step has been considered by some to be androgen synthesis [17, 18], whereas others have considered it to be estrogen formation by aromatization [19, 20]. Most studies, e.g., of porcine [21–24] and bovine [25–27] preovulatory follicles, lack the most relevant information because neither transcript levels nor immunodetectable protein levels provide quantitative estimates of enzyme activity, precluding meaningful comparisons. Enzyme activity is likely to be more directly indicative of steroid synthesis potential because the activities of P450c17 (17,20-lyase) and P450arom (aromatase) are dependent and/or influenced by redox partner protein support [6, 28]. Although the preovulatory estrogen rise might be common among mammals, the regulation of sex steroid synthesis exhibits important species-specific characteristics [6]. For instance, substrate preferences for androgen synthesis are not necessarily the same; 17{alpha}-hydroxyprogesterone is a poor substrate for androstenedione synthesis by bovine (and human) P450c17 but is a good substrate for the porcine enzyme [6]. The pig is even more unusual with respect to estrogen synthesis. It has at least three genes encoding distinct tissue-specific isozymes of P450arom [29, 30], whereas no other mammalian species has yet been found to have more than one complete functional gene, regardless of the tissue site of expression. Additionally, in the pig ovary, the gonad-specific isozyme [31] is expressed prominently in both the follicular granulosa and theca interna [21], but in most other species this isozyme is expressed in granulosa only. This difference provides an opportunity to assess and compare directly the rates of androgen and estrogen synthesis in the same ovarian tissue compartment, as has been done for the interstitial compartment of the porcine testes [32].

The current studies were conducted to evaluate the enzymatic limits to estrogen production from preovulatory porcine follicles by quantifying the relative enzymatic capacity for androgen (17,20-lyase) and estrogen (aromatase) synthesis in theca interna and granulosa compartments. In addition, the support of P450-mediated steroid synthesis by the redox partner flavoprotein reductase was assessed, and the levels and the possible developmental regulation of reductase and cytochrome b5 and of the P450c17 and P450arom proteins was explored.


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

Procedures for handling all animals in this study complied with those specified in the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching [33] and were approved by the MARC Animal Care Committee and the Animal Use and Care Administrative Advisory Committee of the University of California at Davis. Crossbred (Landrace x Yorkshire) primiparous sows were removed from their litters at 16–20 (18.0 ± 0.3) days of lactation. Beginning on the day after weaning, sows were observed twice daily in the presence of a mature boar for occurrence of estrus. Sows were slaughtered from 3 days after weaning through 48 h after estrus to obtain ovarian follicles from the early ovulatory stages through development. Diameter of the largest follicles from each sow (<15/sow) was recorded. Some follicles were dissected free of ovarian stroma and immediately fixed, and the remainder were further microdissected to isolate theca and granulosa cell layers. Sows were grouped based on the combined assessment of mean follicle diameter, follicular fluid estradiol concentration, and behavior: early follicular if not yet in estrus (n = 3) and for those sows that expressed estrus, preovulatory but prior to the ovulatory release of LH (pre-LH; n = 5) and preovulatory but after the LH surge (post-LH; n = 9). These criteria for classification were established in earlier studies [21, 34]. Two sows had already begun ovulation at the time of slaughter and were excluded from the analysis.

Microsome Preparation

Follicular fluid was harvested and pooled within sows, and granulosa cells and theca tissues were isolated as described previously [21] from the largest follicles on both ovaries (10–12/sow), pooled within each sow, and frozen on dry ice. Cells and tissues from each pool were homogenized on ice in buffer (0.1 M potassium phosphate, pH 7.4, 20% glycerol, 5 mM ß-mercaptoethanol, and 0.5 mM PMSF) at a ratio of approximately 1 ml buffer:0.1 g tissue. Microsomes were enriched by subcellular fractionation as previously described [10, 13]. Following brief sonication, cellular debris and mitochondria were removed by centrifugation at 15 000 x g for 10 min. The supernatant was removed and subjected to further centrifugation at 100 000 x g for 60 min, and the pellet was resuspended in homogenization buffer containing 1 mM 3-[3-cholamidopropyl-dimethylammonio]-1-propanesulfonate (CHAPS). Microsomal protein concentration was determined using the Bicinchoninic Acid Protein Assay Reagent (Pierce, Rockford, IL). Aliquots of 100 µg were saved at -80°C for determination of total P450 concentration and steroidogenic enzyme expression and activity. The purity of microsomal fractions was demonstrated previously by immunoblot analysis for microsomal and mitochondrial proteins [13].

Microsomal Enzyme Activities

The 17,20-lyase activity of P450c17 was measured radiometrically [35] as recently described and validated [13]. The assay is based on the release of 3H-acetic acid from [21-3H]-17{alpha}-OH-pregnenolone (25.9 Ci/µmol, prepared in the laboratory of A.M.B.). Microsomal protein (100 µg) was incubated at 37°C in assay buffer (50 mM KPO4, 1 mM EDTA, and 1 mM CHAPS; final volume of 1 ml) in the presence of 10.5 µM 17{alpha}-OH-pregnenolone (7 µM of the radiolabeled and 3.5 µM of unlabeled 17{alpha}-OH-pregnenolone; Steraloids, Wilton, NH) to saturate 17,20-lyase activity [36]. A generating system consisting of 17 mM glucose-6-phosphate, 1 mM NADPH, 2 mM NADP, and 1 U glucose-6-phosphate dehydrogenase (Sigma, St. Louis, MO) was added to maintain a constant supply of reducing equivalents (NADPH). Linearity with time and protein has been reported previously [13], and all subsequent reactions used 100 µg of protein and an incubation time of 2 h. Aromatase activity was assessed similarly in microsomal protein (100 µg) by monitoring the incorporation of tritium from [1ß-3H]-androstenedione (24.7 Ci/mmol; New England Nuclear, Wilmington, DE) into 3H2O as previously described [37]. Microsomal protein was incubated for 2 h at 37°C in the presence of 150 nM androstenedione (20% labeled, 80% cold), exceeding the estimated Km of gonadal P450arom by 1.5-fold [37]. A generating system for NADPH was also added as for the 17,20-lyase assay. These same substrate concentrations and assay conditions were used recently to demonstrate a predominance of aromatase activity over that of 17,20-lyase in human luteinized granulosa cells [38]. In both assays, the incubations were stopped with 30% trichloroacetic acid and extracted with cholorform, and the aqueous phase was combined with a suspension of 8.5% charcoal and 0.85% dextran (17,20 lyase activity) or 5% charcoal and 0.5% dextran (aromatase activity). Following centrifugation at 2000 x g for 30 min, a portion was removed and quantified by liquid scintillation counting. Microsomal reductase activity was measured spectrally by monitoring reduction of cytochrome c at 550 nm as previously reported [10]. Horse heart cytochrome c (40 µM; Sigma) was combined with 100 µM NADPH and brought up to 1 ml in 0.3 M potassium phosphate (pH 7.4). After calibration with a blank at 550 nm, microsomes were added and the absorbance was monitored for 5 min. The activity was calculated as {Delta}A550/min ÷ 0.021 = nmol cytochrome c reduced/min.

Western Immunoblot Analysis

Microsomal proteins (10 µg) were separated by electrophoresis by 8% (P450arom and reductase) or 16% (P450c17 and cytochrome b5) SDS-PAGE in electrode buffer (50 mM Tris, 383 mM glycine, 0.1% SDS, and 0.4 mM EDTA). Separated proteins were transferred onto polyvinylidene fluoride membranes (Immobilon P; Millipore Corp., Bedford, MA). P450arom was detected with a polyclonal antibody raised against recombinant human P450arom (courtesy of Dr. N. Harada, Fujita Health University, Aichi, Japan) at 1:2000 dilution. P450c17 was detected with a 1:2000 dilution of an antiserum raised against purified porcine P450c17 (gift of Dr. A. Payne, Stanford University Medical Center, Stanford, CA). Reductase was detected with a 1:5000 dilution of an antiserum raised in our laboratory against purified recombinant P450 reductase protein. Cytochrome b5 was detected with a 1:5000 dilution of antiserum similarly raised in our laboratory using purified recombinant human protein generously provided by Drs. Ron Estabrook and Manju Shet (Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX). Immunoblotting procedures were carried out at room temperature in PBS with 0.1% Tween 20 (Amresco, Solon, OH). After blocking in 20% dried milk, the proteins were visualized by incubating the membranes with donkey anti-rabbit horseradish peroxidase–linked IgG whole antibody (Amersham, Arlington Heights, IL) at 1:10 000 dilution, washing, and then generating a chemiluminescent signal using luminol reagent (New England Nuclear). Following autoradiography, the immunodetectable bands were quantified by densitometry.

Immunocytochemistry

Representative follicles from pre-LH and post-LH sows were fixed in 4% paraformaldehyde at 4°C for 24 h and processed as previously described [39]. Immunocytochemical localization of P450c17, P450arom, and cytochrome b5 was performed using the avidin-biotin-peroxidase complex method with the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA). Sections were deparaffinized using CitriSolv (Fisher Health Care, Houston, TX), hydrated through a graded alcohol series, and rinsed in water. Endogenous peroxidase activity was then blocked in 0.3% H2O2 for 30 min followed by washing in buffer. Antigen retrieval was conducted for P450arom only by heating the sections to 95°C for 25 min in citrate buffer (pH 6.0) using a rice steamer in which temperature was monitored continuously. Slides were blocked in dilute normal goat serum for 20 min and then incubated for 16 h at 4°C with the primary antibody. P450arom and cytochrome b5 were detected using the antibodies described above at dilutions of 1:1500 and 1:10 000, respectively. P450c17 was detected with an antiserum raised in our laboratory against purified recombinant bovine P450c17 protein at 1:3000. Dilute biotinylated goat anti-rabbit IgG secondary antibody was applied for 30 min followed by a 30-min incubation with the avidin-biotin-peroxidase complex. Visualization was achieved using the AEC Substrate kit (Vector). Slides were counterstained with hematoxylin and mounted using an aqueous mounting medium. Negative controls using preimmune rabbit serum were processed for both steamed and nonsteamed sections.

Hormone Assays

Follicular fluid samples were diluted in buffer and assayed without extraction for estradiol-17ß and progesterone, in a single assay for each, using [125I]-labeled tracers as previously described [13, 40]. The >=10-fold dilutions required to hit the standard curves of both assays obviated any need for extraction. Intraassay coefficients of variation were 15% for estradiol-17ß and 11% for progesterone.

Statistical Analysis

Data were evaluated by least-squares ANOVA using the general linear models procedures of the SAS Institute [41]. Data were log transformed when heterogeneity of variances were encountered but are presented here as least squares means ± SEM. When an F-test was significant, specific means were compared with Tukey test results. Simple correlations were determined by regression analysis.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The follicles collected represented a wide range of preovulatory development and steroidogenesis based on both follicular diameter and antral fluid steroid concentrations that were nonsignificantly unrelated to the day of weaning when assessed in covariate analyses (data not shown). As defined, estradiol concentrations in estrous sows were highest in follicular fluid from the pre-LH group and were lowest in the post-LH group (P < 0.01). Progesterone was lower in follicular fluid from the early follicular animals than that from either the pre-LH or post-LH groups (P < 0.05). Changes in antral fluid steroid concentrations accompanied a near doubling of follicular diameter from early follicular to post-LH animals (P < 0.05; Table 1). Aromatase activities in microsomes from theca and granulosa were correlated (P < 0.01) with the log of follicular fluid estradiol concentrations (Fig. 1). Granulosa aromatase activities were higher than those of theca aromatase (P < 0.01). Thecal 17,20-lyase activity was also correlated with that of estradiol in follicular fluid (P < 0.01; Fig. 1). 17,20-Lyase activity peaked in pre-LH sows and fell dramatically, along with aromatase activity, in theca and granulosa of post-LH animals. Results of immunoblot analyses were consistent with these data. Aromatase and 17,20-lyase activities were highly correlated with immunodetectable levels of P450c17 (Fig. 2) and P450arom (Fig. 3) protein, with correlation coefficients (R2) ranging from 0.71 to 0.82 (P < 0.05). By comparison with theca, P450c17 was essentially undetectable in the granulosa of most animals (Fig. 2), and immunodetectable levels of P450arom were always higher in the granulosa (Fig. 3). Background was low on all immunoblots, and both P450c17 (50 kDa) and P450arom (48 kDa) were detected as single clear bands of the expected molecular sizes. More importantly, however, granulosa and theca aromatase activities were always markedly lower in absolute terms than thecal 17,20-lyase activity, by one or two orders of magnitude (Table 2), regardless of the stage of follicular maturation.


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  		TABLE 1. Porcine follicular diameter and antral fluid steriod concentrations (least squares means ± SEM) by stage of preovulatory follicular development



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  		FIG. 1. Correlations of enzyme activities and antral fluid estradiol concentrations in porcine preovulatory follicles. Microsomes were isolated from the theca and granulosa. Theca aromatase ({blacksquare}), granulosa aromatase ({diamondsuit}), and theca 17,20-lyase ({blacktriangleup}) activities were measured and plotted as a function of estradiol concentration. Equations and estimated correlation coefficients (P < 0.01) are shown for each line. Each point represents values obtained from tissues and fluid pooled within individual pigs from the early follicluar, pre-LH, and post-LH groups. The vertical dotted line distinguishes pre-LH from early follicular and post-LH points. Note the different scales representing 17,20-lyase activity (nmol mg-1 2 h-1) and aromatase (pmol mg-1 2 h-1) such that lyase activity actually exceeds aromatase activity by 2 orders of magnitude.



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  		FIG. 2. Western immunoblot analysis to detect P450c17 and cytochrome b5 expression in theca and granulosa compartments of porcine preovulatory follicles. Microsomal proteins (10 µg/lane) were isolated from theca tissues (T) and granulosa cells (G) of follicles pooled within individual pigs. Follicular fluid estradiol (E2) concentration is shown at the bottom for each of the three animals shown, and molecular size markers are shown on the right. An equal amount of microsomal protein from porcine neonatal testes (far right lane) was included as a positive control



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  		FIG. 3. Western immunoblot analysis to detect P450arom and reductase expression in theca and granulosa compartments of porcine preovulatory follicles. Microsomal proteins (10 µg/lane) were isolated from theca tissues (T) and granulosa cells (G) of follicles pooled within individual pigs. Follicular fluid estradiol (E2) concentration is shown at the bottom for each of the three animals shown, and molecular size markers are shown on the right. An equal amount of microsomal protein from porcine neonatal testes (far right lane) was included as a positive control


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  		TABLE 2. Porcine theca and granulosa enzyme activities (least squares means ± SEM) in microsomal fractions by stage of preovulatory follicular development

In contrast to aromatase and 17,20-lyase, reductase activities did not change with the stage of follicular maturation. However, reductase activities in the granulosa were consistently twice those in the theca (P < 0.01; Table 2), and immunoblot analysis confirmed that theca expressed lower reductase activity (Fig. 3). Cytochrome b5 was detectable in both theca and granulosa microsomes by immunoblot analysis, but in contrast to reductase, activity was always higher in theca (Fig. 2). Expression of cytochrome b5 was also positively correlated with P450c17 expression (R2 = 0.45) and therefore appeared to be higher in the theca and granulosa of pre-LH animals. However, correlations with 17,20-lyase activity were much lower (R2 = 0.24), and there was no correlation between cytochrome b5 expression and aromatase activity (R2 = 0.004). For P450c17 and P450arom, single bands were detected for both reductase (70 kDa) and cytochrome b5 (16 kDa) at the expected molecular sizes.

Immunocytochemistry was used to verify the tissue-specific expression of P450c17, P450arom, and cytochrome b5 (Fig. 4). P450arom was detected in pre-LH follicles and was diffuse and moderately positive within the granulosa with a lower multifocal expression evident within the theca interna (Fig. 4A). All other cells types were negative. P450arom was minimal to negative within post-LH follicles (Fig. 4B). The expression of P450c17 was intense and diffusely positive within the theca interna of both pre-LH and post-LH follicles and was not detected in the granulosa or other cell types (Fig. 4C). Cytochrome b5 was intense and diffusely positive within the granulosa and theca interna of pre-LH follicles (Fig. 4D). Within post-LH follicles, cytochrome b5 cell-specific expression was similar to that seen in pre-LH follicles; however, expression in the granulosa cell layer was less intense. In addition, the granulosa cell layer of adjacent developing primary and small secondary follicles were moderately and diffusely positive for cytochrome b5 expression, whereas the granulosa of large secondary and small tertiary follicles were negative. The surface mesothelium and the theca interna surrounding these small tertiary follicles were strongly positive for cytochrome b5 expression. All other cell types were negative. Replacement of the primary antiserum with preimmune rabbit serum eliminated staining in all cases (data not shown), resembling the lack of staining for P450arom in post-LH follicles (Fig. 4B).



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  		FIG. 4. Immunolocalization of steroidogenic enzymes and cytochrome b5 in the wall of preovulatory porcine follicles before (pre-LH) and after (post-LH) the presumed surge of LH. A) P450arom expression in follicle wall from a pre-LH sow. Strong expression is visible diffusely throughout the granulosa cell layer (G), and lower intensity multifocal staining is visible within the theca interna (T), along with occasional strongly positive T cells (arrowhead). B) P450arom expression in a preovulatory post-LH follicle. Note the minimal staining within both the G and T. C) P450c17 expression in a preovulatory follicle from a pre-LH sow. There is minimal staining within the G in contrast to intense and diffuse staining within the T. D) Cytochrome b5 expression within a pre-LH preovulatory follicle, showing intense diffuse staining within the G and T. Bar = 10 µm


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The primary goal of this study was to quantify directly and compare the rates of androgen (17,20-lyase) and estrogen (aromatase) synthesis. Despite correlations between 17,20-lyase, aromatase, and follicular fluid estradiol concentrations, 17,20-lyase activity always greatly exceeded aromatase activity, by >2 orders of magnitude (>100-fold) at the peak of follicular fluid estradiol accumulation. These differences are consistent with estimates made by alternative methods. Androgen concentrations (testosterone + androstenedione) were 25-fold higher than estrogen (estradiol only) concentrations in cultures of porcine theca and granulosa [42], close to 100-fold higher when estimated on a per follicle basis [43]. Wide ranges have been reported for androgen and estrogen concentrations in utero-ovarian venous plasma of pigs, but androgens generally exceeded estrogens, by 10-fold in one study [44], although a smaller difference was seen between testosterone and estradiol in another study [40]. Total androgen or estrogen synthesized was not determined in these studies, and no matter how close to the ovary the samples are collected, the determinations still fail to account for steroid concentrations in lymph, which generally are much higher [45, 46]. Follicular fluid steroid concentrations also do not necessarily reflect concentrations in ovarian venous blood, at least in pigs [47]. Grant et al. [48] found that follicular fluid testosterone and estradiol concentrations rose and fell in concert, although testosterone concentration in theca tissues continued to rise in pig follicles on Days 16–21 of the estrous cycle. Based on estradiol concentrations in utero-ovarian venous plasma, which peaked 4 days after a luteolytic dose of prostaglandin F2{alpha} [49], it is more likely that follicular estradiol secretion into blood is already near maximal in the early follicular stages, as were theca and granulosa P450arom activities, even though this peak was not yet evident as increases in follicular fluid estradiol concentrations. Discrepancies between antral fluid and ovarian venous plasma steroid concentrations may be due to a greater contribution estrogen output from the theca into circulation, because this layer is heavily vascularized. Conversely, estrogen from the granulosa may move primarily into the follicular fluid because of the presence of the high-capacity low-affinity binding by albumin [50]. Regardless of the mechanisms involved, follicular fluid steroid levels do not reflect the rates of secretion as accurately as do enzyme activities, and 17,20-lyase activity far exceeds the rate of aromatization in the follicle wall at the peak of estrogen secretion.

The radiometric methods used to compare the rates of C-19 and C-18 steroid synthesis were not dependent on the identification and summation of specific steroid products, presumed to be the quantitatively major metabolites, in different fluid compartments. Results of immunocytochemistry confirmed that P450arom [51] and P450c17 [39] were expressed in the theca interna [51] and that cytochrome b5 was expressed in the granulosa, as suggested by immunoblot analysis. These data indicate that contamination of theca and granulosa compartments during follicle dissection was most likely minimal. Even though any method will provide only an approximation of enzyme activity, given the magnitude of differences between 17,20-lyase and aromatase noted here, two conclusions can be reached. First, it seems unlikely that the capacity for aromatization by theca and granulosa compartments could ever exceed the rate of androgen supply from the porcine theca interna and therefore must ultimately limit the rate of estrogen formation. This is not true of human luteinized granulosa cells, in which aromatase activity exceeds that of 17,20-lyase by 10-fold and 17,20-lyase, not aromatase, limits estrogen synthesis [38, 52, 53]. Diffusion of steroid across the basement membrane separating the theca from the granulosa may decrease the apparent disparity between androgen supply in one follicle compartment and the rate of aromatization in the other, which is the case in most species. However, in pigs P450arom is expressed in the theca interna along with P450c17 [21]; thus, no diffusion gradient exists, and the differential between androgen and estrogen synthesis must serve other needs. Second, given the disparity between the rates of androgen synthesis and aromatization, it is equally unlikely that changes in the rate of aromatization could influence C-19 steroid secretion by the ovary because the capacity for metabolism by this route represents such a small fraction of total steroid synthesis. Therefore, even though uniquely positioned in the steroidogenic pathway to affect the balance of androgens and estrogens in tissues, P450arom limits and therefore regulates estrogen synthesis at a level that allows independent regulation of androgen output.

An additional aim of the present experiments was to evaluate redox partner, i.e., reductase and cytochrome b5, protein expression in relation to P450c17 and P450arom and to evaluate possible regulatory roles in follicular steroidogenesis. Although reductase has been examined by immunocytochemistry in bovine follicles [54], we are unaware of any previous reports of activities or of any assessment of cytochrome b5 expression in ovarian tissues. Reductase activities in the granulosa were twice those in the theca. Both were in the range obtained in developing porcine testes [13], although testes have much higher activities of total microsomal P450 (unpublished results) [10]. In contrast to neonatal testes [13], in which reductase activity changed several fold during postnatal growth, reductase activity did not vary with 17,20-lyase or aromatase activities in either granulosa or theca compartments. Therefore, it seems unlikely that reductase activity could ever limit microsomal P450-mediated steroid synthesis in the porcine follicle, although this can apparently occur in vitro [10]. The opposite circumstance appears to hold for cytochrome b5. Cytochrome b5 expression did not appear to change during neonatal testicular development, but in the ovary, it increased with both follicle maturation and follicular fluid estrogen concentration and was correlated (R2 = 0.45) positively with the expression of P450c17 in the theca interna. Although cytochrome b5 supports 17,20-lyase activity [14] and is highly expressed in the porcine testes [13, 55], correlations with 17,20-lyase activity were much lower (R2 = 0.24). These data suggest that P450c17 and cytochrome b5 expression may be regulated coordinately but that cytochrome b5 concentration is not likely to be a physiological regulator of 17,20-lyase activity. This conclusion is consistent with the finding of cytochrome b5 in granulosa that had no detectable P450c17 expression [21] or 17,20-lyase activity (data not shown). Because most steroidogenic tissues with prominent cytochrome b5 expression generally exhibit the capacity for androgen synthesis [56, 57] (unpublished results), this level of expression was surprising. Despite these apparently high concentrations and the report that human P450arom can be inhibited by cytochrome b5 [16], there was no correlation between cytochrome b5 expression and granulosa aromatase activity (R2 = 0.004). In sum, no evidence was found that supported direct regulatory roles for either reductase or cytochrome b5 in P450-mediated follicular estrogen synthesis. Additionally, the expression of these redox partner proteins appears itself to be regulated by different mechanisms in these tissues.

The differences between ovarian and testicular gametogenesis are well recognized and described [58], but few comparisons have been made relative to the capacity for sex steroid synthesis. Concentrations of androgens exceed those of estrogens by 5- to 20-fold in testicular venous plasma [59, 60], not unlike the differences seen in ovarian venous plasma [40, 44]. As estimates of rates of steroid synthesis, these findings are consistent with the relative 17,20-lyase and aromatase activities reported here and previously in porcine neonatal testes [13] at a stage of development when tubular volume is low and interstitial volume is at its highest [61]. Although more variable among follicles, the highest theca 17,20-lyase activity (62.2 nmol mg-1 2 h-1) was close to the highest mean activity found in neonatal testes (80 nmol mg-1 2 h-1); aromatase activity was the same in follicles and testes (0.30 nmol mg-1 2 h-1). Reductase activity varied greatly in developing testes but was comparable nonetheless (50 nmol mg-1 min-1 in theca and 20–100 nmol mg-1 min-1 in testes). Immunoblot analysis indicated that cytochrome b5 concentration also was similar in theca and testes (Fig. 2). Thus, based on microsomal protein concentations and enzyme activities, there are few major differences in the capacity for androgen or estrogen synthesis by preovulatory theca and testicular interstitial tissue, despite disparities in total P450 content. Differences between follicular and testicular steroidogenesis are more apparent in the types of androgens synthesized. In boars, dehydroepiandrosterone, dehydroepinadrosterone sulfate, 5{alpha}-androst-16-ene-3-one, and testosterone [59, 60, 62, 63] are the major secreted androgens. Although androgens have not been studied extensively in females, a variety of steroids have been detected in porcine follicular fluid [64]. Androstenedione exceeded testosterone synthesis in cultured porcine theca [42, 43] and in utero-ovarian venous plasma by 2- to 20-fold [44]. Therefore, differences in tissue mass, the types of androgens and estrogens secreted, and the relatively short interval during which ovarian steroidogenesis reached levels seen in the testes likely contribute most to the differences in circulating sex steroids and their effects in males and females. The stimulation of ovulation rate in pigs by testosterone [65, 66] and dihydrotestosterone [67] and the expression androgen receptor in porcine granulosa [23, 68] suggest that androgens play an important role in the physiology of follicle growth and development in pigs.

Cytochrome b5 is best known to steroid biochemists for its ability to support the 17,20-lyase activity of P450c17 [69, 70] and therefore to promote, or if absent preclude [14], androgen synthesis in tissues expressing P450c17 [6]. However, the role of cytochrome b5 as a modulator of 17,20-lyase activity through electron transfer has been questioned [28, 71]. The prominent expression of cytochrome b5 in the granulosa of pigs, where little or no P450c17 expression is found, suggests that this protein may have other specific functions in steroidogenic cells and possibly in other androgen-secreting tissues. There was no correlation between 17,20-lyase activity and cytochrome b5 concnetration even in theca in this study nor were changes in 17,20-lyase activity associated with alterations of cytochrome b5 concentrations in neonatal testes [13]. Neonatal testes and theca exhibited similar 17,20-lyase and P450c17 (and P450arom) activities, but whereas testicular microsomes had readily detectable total P450 concentrations [13], total P450 concentrations were detectable in only three sows in the present study. This further supports the conclusion that microsomal steroid hydroxylases contribute relatively little to total P450 concentrations in gonadal tissues. Therefore, cytochrome b5 may support the activities of other microsomal P450 in neonatal testes, if not also the theca and granulosa of ovarian follicles. These P450s, their major activities, and physiological relevance remain to be identified but may contribute to differences in testicular and ovarian endocrine function in an important way.


   ACKNOWLEDGMENTS
 
The authors thank Todd Boman for help with steroid analysis and the personnel of the USMARC Swine Operations unit for assistance with the care and maintenance of animals. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.


   FOOTNOTES
 
1 This work was supported by grant USDA-NRI 98-35203-6439 (to A.J.C). J.D.V. was supported by training grant funds (ES07055) from NIEHS. Back

2 Correspondence: Alan Conley, Department of Population Health & Reproduction, School of Veterinary Medicine, 1131 Tupper Hall, University of California, Davis, CA 95616. FAX: 530 52 4278; ajconley{at}ucdavis.edu Back

Received: 5 February 2003.

First decision: 25 February 2003.

Accepted: 12 March 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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