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Endocrinology Vol. 143, No. 9 3361-3369
Copyright © 2002 by The Endocrine Society

ARTICLE

Regulation of Microsomal P450, Redox Partner Proteins, and Steroidogenesis in the Developing Testes of the Neonatal Pig

F. M. Moran, J. J. Ford, C. J. Corbin, S. M. Mapes, V. C. Njar, A. M. Brodie and A. J. Conley

Department of Population Health & Reproduction (F.M.M., C.J.C., S.M.P., A.J.C.), School of Veterinary Medicine, University of California, Davis, California 95616-8743; United States Department of Agriculture (J.J.F.), Agricultural Research Service, Roman L. Hruska United States Meat Animal Research Center, Clay Center, Nebraska 68933; and Department of Pharmacology & Therapeutics (A.M.B., V.C.N.), University of Maryland School of Medicine, Baltimore, Maryland 21201-1559

Address all correspondence and requests for reprints to: Alan J. Conley, University of California at Davis, Veterinary Medicine—Population Health and Reproduction, School of Veterinary Medicine, Davis, California 95616-8743.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Testicular growth and plasma androgen concentrations increase markedly in the first weeks of neonatal life of pigs. The regulation of steroidogenesis through this period was examined by measuring total microsomal cytochromes P450 (P450), 17{alpha}-hydroxylase/17,20-lyase P450 (P450c17) and aromatase P450 (P450arom) enzyme activities, and the redox partner proteins nicotinamide adenine dinucleotide phosphate, reduced form (NADPH)-cytochrome P450 reductase (reductase) and cytochrome b5 in testicular microsomes. Testes were collected from 1–24 d of age, and testicular development was suppressed by a GnRH antagonist in some animals from d 1–14. Both 17/20-lyase and aromatase activities increased from d 1–7 but not thereafter, and 17–20-lyase activity was always at least 200-fold higher than aromatase activity. Reductase decreased in wk 1, then increased to d 24. No changes were seen in cytochrome b5 expression. GnRH antagonist treatment suppressed plasma LH, testosterone and testes growth to d 14. 17,20-Lyase and aromatase activities in testicular microsomes were reduced by 20% and 50%, respectively. Total microsomal P450 concentration was reduced by 50% on d 7, but there was no effect of treatment on reductase or cytochrome b5 expression. These data support the hypothesis that the rise in neonatal testicular androgen secretion is more likely due to gonadotropin-stimulated gonadal growth, rather than specific P450c17 expression. Neither P450c17 nor P450arom can account for the decline in total microsomal P450. Reductase and cytochrome b5 expression appears to be constitutive, but reductase levels saturate both P450c17 and P450arom.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE DOMESTIC BOAR, like the stallion, is unusual among mammals in having high circulating concentrations of estrogens (1, 2) compared with either preovulatory females of the same species (3), or the males of other species based on urinary secretion (4). Levels of free androgens (5), estrogens (6), and conjugated steroids (7) are particularly dynamic in the early neonatal period. Steroid concentrations reach a peak in the first month of life, decline transiently, then increase again as puberty is initiated, because of a decrease in clearance, as well as an increase in steroid production (8). The interstitial compartment comprises the majority of testicular volume in the early neonatal pig (9, 10), is the site of expression of steroidogenic enzymes in the boar (11, 12), and therefore the major cellular site of both androgen and estrogen synthesis. Leydig cell development and function (13) is dependent on pituitary support in late gestation (14) and postnatally (15, 16, 17, 18, 19, 20, 21). The compensatory increase in testicular interstitial and tubular spaces, and total mass, which is seen following unilateral castration in the neonatal period (10, 22, 23), is associated with a sustained increase in plasma FSH, but not testosterone or LH concentrations (24). In contrast to the extensive studies conducted on laboratory rodents (13, 25, 26), relatively little is known of the acute effects of gonadotropic stimulation in vivo on the components of the testicular steroidogenic apparatus of pigs beyond peripheral steroid concentrations.

The capacity of the testicular interstitium of pigs to elaborate significant quantities of both androgens and estrogens may also be of biochemical interest because the requisite enzyme activities are more often compartmentalized than not in reproductive systems. This is most obvious in the ovaries for instance, wherein androgen synthesis occurs in the theca and estrogen production from androgens takes place in the granulosa of preovulatory follicles (27, 28). This is true of all mammals studied to date, with the possible exception of the pig in which the theca expresses the enzymes (29) and activity (30) consistent with estrogen formation from androgens. Androgen (C-19 steroid) and estrogen (C-18 steroid) synthesis is catalyzed by the microsomal cytochrome P450 enzymes, 17{alpha}-hydroxylase/17,20-lyase (P450c17) (31, 32) and aromatase (P450arom) (33, 34), respectively. Like all microsomal P450 enzymes, both P450c17 and P450arom are functionally dependent on the microsomal redox partner, flavoprotein nicotinamide adenine dinucleotide phosphate, reduced form (NADPH)-cytochrome P450 oxidoreductase (reductase) for their activity (35). Additionally, reductase (36), and another potential redox partner protein, cytochrome b5 (32, 37, 38, 39, 40), may be involved in selectively regulating the level of 17,20-lyase activity of P450c17 (41). The porcine testes is known to be a rich source of both reductase and cytochrome b5 (36, 38, 42), but little is known concerning the regulation of the expression of either protein in steroidogenic tissues of this or any other species. Moreover, the potential exists for competition between P450c17 and P450arom for reductase binding in the microsomal membrane of steroidogenic cells, at least in the testicular interstitium, and this might influence the efficiency of either androgen or estrogen synthesis. It is known for instance that microsomal P450s expressed in the liver compete (43) for a limited pool of available reductase (44, 45). However, the significance of competition between P450c17 and P450arom for reductase in cells in which these two enzymes are coexpressed will be determined by multiple factors including the level of redox partner protein expression, as well as the relative levels of the P450s themselves. To our knowledge, these factors have not been evaluated previously in reproductive tissues, either male or female, of any species.

Therefore, the following study was conducted to examine the expression of microsomal P450s, reductase, and cytochrome b5, and the activities of P450c17 and P450arom, that catalyze androgen and estrogen synthesis during the dynamic, neonatal period of growth in pigs. In addition, testicular development was inhibited by suppression of pituitary gonadotropin release through the first 2 wk of life. It was hypothesized that the expression and activities of steroidogenic enzymes and redox partner proteins would be suppressed, along with testicular growth, by a lack of gonadotropic hormonal support.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Sixteen newborn piglets (white composite line) received a daily im injection of vehicle (n = 7; saline/control) or were treated with the GnRH antagonist, SB75 (50 µg/kg body weight/d; n = 9), as reported previously (19). Animals were examined at slaughter after either 7 (n = 3 control, n = 4 treated) or 14 d (n = 4 control, n = 5 treated) later. Ten additional untreated animals were examined on 1 (n = 5) and 24 (n = 5) d of age. At slaughter, testes were removed, weighed, and stored at -80 C until used for subsequent analyses. In a separate experiment, testis were also collected from 3- (n = 8) and 60-d-old (n = 10) piglets and processed similarly. 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 (46).

Microsome preparation and validation
Tissue from each individual testis was homogenized on ice in buffer (0.1 M potassium phosphate, pH 7.4; 20% glycerol; 5 mM ß-mercapto-ethanol) with the protease inhibitor phenylmethylsulfonyl fluoride (0.5 mM) at a ratio of 1 ml buffer/0.1 g tissue. Microsomal fractions were prepared by subcellular fractionation (47). Cellular debris was removed by centrifugation at 1,000 x g for 10 min, and a premicrosomal pellet was obtained after centrifugation at 15,000 x g for another 10 min. The supernatant was centrifuged at 100,000 x g for 60 min and the pellet, containing enriched microsomes, was resuspended in 0.1 M KPO4, 20% glycerol, 5 mM ß-mercapto-ethanol and 1 mM 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS). Several protocols for the preparation of microsomes from liver (48, 49) include a salt wash (0.15 M KCl) to reduce hemoglobin contamination that might interfere with spectral determinations. Preliminary studies with porcine testes microsomes showed that the inclusion of a salt wash did not alter estimates of P450 concentration made, perhaps because they suffer less red blood cell contamination than do liver specimens. This step was not included in the preparation of microsomes from the testes of the animals in the study. Microsomal protein concentration was determined using the bicinchoninic acid protein assay reagent (Pierce Chemical Co., Rockford, IL) with BSA as standard. Aliquots of 100 µg were saved at -80 C for total P450 concentration as well as steroidogenic enzyme expression and activity determinations.

The purity of microsomal fractions was evaluated by immunoblot analysis as described below. Equal amounts (10 µg) of premicrosomal and microsomal protein fractions were serially diluted (1:2, 1:4, and 1:8) with buffer, subjected to electrophoresis and immunoblotted for mitochondrial cholesterol side-chain cleavage cytochrome P450 (P450scc) and microsomal cytochrome b5 (Fig. 1Go). P450scc was clearly detectable in the premicrosomal fraction, even after being diluted 8-fold. P450scc was detectable in the microsomal fraction, but the level without dilution was lower than the most diluted premicrosomal fraction (Fig. 1Go, upper panel). In addition, cytochrome b5 though detectable in the undiluted, premicrosomal fraction was obviously lower than the microsomal fraction after it had been diluted 8-fold (Fig. 1Go, lower panel). These analyses indicated that the recovery of microsomal protein was probably 90% or better, and that microsomal contamination with the premicrosomal fraction was likely less than 10% of the mitochondria-containing protein pool.



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  		Figure 1. Immunoblot analysis of premicrosomal and microsomal proteins (10 µg) prepared from porcine testes microsomes. The upper and lower panels show the results of analyses for cholesterol side chain cleavage cytochrome P450 (P450scc) and cytochrome b5, respectively. Note that both the premicrosomal and microsomal fractions have been diluted as shown below (1:2, 1:4 and 1:8 before loading). The molecular size estimate is shown on the right. Note that though P450scc can be detected in the microsomal fraction, the level is lower than the highest dilution of the premicrosomal fraction. Similarly, though cytochrome b5 is detected in the premicrosomal fraction, the level is well below the highest dilution of the microsomal fraction. The protein control (C) is testes homogenate.

 
Total microsomal P450
Total cytochrome P450 concentration was measured by CO difference spectra (Fig. 2Go) using the sodium dithionite-reduced sample, as originally described (50). Briefly, microsomal samples were diluted and split into two glass cuvettes. After a baseline was recorded, CO was bubbled into the sample cuvette; a few grains of sodium dithionite (Fluka, Milwaukee, WI) were added to both cuvettes, mixed and absorbance recorded using a split-beam spectrophotometer (UV-2401PC, Shimadzu Co., Pleasanton, CA) coupled to an IBM compatible computer to capture the data. The absorbance was recorded from 400–500 nm over several minutes (Fig. 2Go). Total P450 concentration for each sample was obtained from the equation, A450-A490 x 0.091-1 = P450 nmol/ml (48). Similar values were obtained using the equation, A460-A490 x 0.052-1= P450 nmol/ml, as suggested by Kowal et al. (51) to minimize error introduced by potential contamination with mitochondrial pigments. Therefore, data are reported based on A450-A490 x 0.091-1.



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  		Figure 2. Difference spectra were recorded on testicular microsomes in a dual beam spectrophotometer to quantify total P450 concentration according to Omura and Sato (50 ). Microsomal fractions (1 ml) were diluted 1:2 in buffer, split into 2 x 1 ml cuvettes (sample and reference), and a baseline was recorded. Carbon monoxide was bubbled into the sample cuvette, a few grains of sodium dithionite were added to both cuvettes, each was mixed and the resulting difference spectrum was recorded between 400 and 500 nm (horizontal scale) over several minutes. The left panel shows a typical spectrum. The development of the P450 peak absorbance (vertical scale) over time can be seen in the sequential tracings shown. The right panel shows an amplification of the area around the 450-nm peak.

 
Enzyme activity
The 17,20-lyase activity of P450c17 was measured radiometrically as described elsewhere, with minor modifications (52). Briefly, the assay is based on the release of 3H-acetate from [21-3H]-17{alpha}-OH-pregnenolone (25.9 µCi/µmol, prepared in the Brodie laboratory). Microsomal protein (100 µg) was incubated at 37 C in assay buffer [50 mM potassium phosphate, 1 mM EDTA, 1 mM 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (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). Generating system, 17 mM glucose-6-phospate, 1 mM NADPH, 2 mM ß-nicotinamide adenine dinucleotide phosphate, and 1 U of glucose-6-phosphate dehydrogenase (Sigma, St. Louis, MO) was added to maintain a constant supply of reducing equivalents (NADPH). The assay validation in terms of protein and time course is depicted in Fig. 3Go. The reaction was linear up to 4 h with 100 µg of microsomal protein. Therefore, all subsequent reactions used 100 µg of protein and an incubation time of 2 h.



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  		Figure 3. Validation of 17,20-lyase activity measurement showing linearity with testis microsomal protein (A: 1, 10, or 100 µg) and time (B: inset shows linearity over 2 h). Details of the assay are described in Materials and Methods.

 
P450arom activity on microsomal protein was assessed by the incorporation of tritium to water from [1ß-3H]-androstenedione (25.9 Ci/mmol, NEN Life Science Products, Wilmington, DE) as previously described (53). Briefly, 100 µg of microsomal protein was incubated for 2 h at 37 C in the presence of 150 nM (20% labeled, 80% cold androstenedione, Steraloids, Wilton, NH) and NADPH generating system as explained above for the 17,20-lyase assay. Microsomal reductase activity was measured spectrally by monitoring reduction of cytochrome-c at 550 nm as previously reported (47). Identical biochemical analyses were conducted on testicular microsomes isolated from the 3- and 60-d-old piglets, and additional studies were conducted to examine the adequacy of reductase support for P450c17 and P450arom. This was achieved by measuring 17,20-lyase and aromatase activities after augmenting available reductase with active recombinant protein at levels that were anticipated to double endogenous reductase activity. Earlier studies using microsomes from cell lines overexpressing porcine P450arom have demonstrated that recombinant reductase addition can stimulate aromatase activity in microsomes severalfold (47). For each activity measured, 17,20-lyase, aromatase and reductase, samples from all animals were run in single assay to circumvent possible interassay variation.

Western immunoblot analysis
Microsomal proteins (10 µg) were separated by electrophoresis on 8% (P450arom and reductase) or 16% (P450c17 and cytochrome b5) SDS-PAGE for 1 h at 150 V in electrode buffer (50 mM Tris; 383 mM glycine; 0.1% sodium dodecyl sulfate; and 0.4 mM EDTA). Separated proteins were transferred onto polyvinylidene difluoride membranes (Immobilon P, Millipore Corp., Bedford, MA) at 30 V for 18 h in transfer buffer. Proteins were detected by immunoblotting the polyvinylidene difluoride membrane with rabbit antisera as a primary antibody. P450arom was detected with an antibody raised against recombinant human P450arom (courtesy of Dr. N. Harada, Fujita Health University, Toyoake, Aichi, Japan) at 1:2000 dilution. P450c17 was detected with a 1:2000 dilution of an antisera 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 antisera raised in our laboratory (54) against 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). Finally, P450scc was detected in premicrosomal and microsomal fractions using antisera raised against human P450scc (gift from Dr. Walt Miller, University of California San Francisco, San Francisco, CA) at a dilution of 1:1000. Immunoblotting procedures were carried out at room temperature in PBS with 0.1% Tween 20 according to the manufacturer’s instructions (electrochemiluminescence, Amersham Pharmacia Biotech, Arlington Heights, IL). Visualization of protein was made possible by incubating the membranes with a donkey antirabbit horseradish peroxidase-linked IgG whole antibody (Amersham Pharmacia Biotech) at 1:10,000 dilution. Immunoreactive bands were visualized by autoradiographic detection of the chemiluminescent signal (NEN Life Science Products). Quantification of each immunoblot was possible by scanning the autoradiographic films and measuring the dot intensity by densitometry.

Hormone assays
Blood samples were obtained by venipuncture from all boars on d 7 and 14. Plasma was stored at -20 C until assayed. Circulating LH levels were determined by RIA as previously reported (55). Briefly, all samples were included in a single assay that used LH antiserum AFP-151031194, porcine LH (AFP-10714B) for iodination and porcine LH-B1 as the reference preparation. Intraassay coefficients of variation ranged from 1.4% to 15.4% for four pools of porcine serum. Testosterone (T4) levels were determined by RIA using commercially available reagents (Diagnostic Systems Laboratories, Webster, TX). All samples were included within a single assay. Sensitivity of the T4 assay was 80 pg/ml and the intraassay coefficient of variation was 11.2%.

Statistical analysis
Developmental changes were evaluated from data collected on d 1 and 24, and from untreated animals on d 7 and 14, by one-way ANOVA. The effects of gonadotropin suppression on these same parameters were evaluated in the same untreated animals and treated contemporaries on d 7 and 14 by two-way ANOVA. Regression analysis was also used to determine correlation coefficients among hormone levels, enzyme activity and testicular weight.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Testicular weight increased (P < 0.001) almost 10-fold in control boars from 0.30 ± 0.03 g, at 1 d of age, to 3.57 ± 0.83 g by d 24 (Fig. 4AGo). Testicular growth from d 1–7 was also associated with an increase in total P450 concentration (Fig. 4BGo; P < 0.05), 17,20-lyase (Fig. 4CGo; P < 0.05), and aromatase activities (Fig. 4DGo; P < 0.05). However, none of these parameters was seen to undergo significant changes thereafter. It is notable also that 17,20-lyase activity was two orders of magnitude higher than aromatase activity, never less than 200-fold higher in fact. In contrast to the lack of changes in P450 levels after d 7, there were significant fluctuations in microsomal reductase activity among testes from all ages (Fig. 5Go). Indeed, the increases in P450 concentration and enzyme activities occurring during the first week of life were associated with a 50% decrease in reductase between d 1 and 7 (from 44.5 ± 10.4 to 21.3 ± 1.1 nmol/mg·2 h). However, this was followed by a steady increase through d 14 (34.6 ± 17.4 nmol/mg·2 h) and 24 (109.9 ± 8.6 nmol/mg·2 h) by which time reductase activity was more than 5-fold higher than that seen on d 7. There was no apparent change in cytochrome b5 levels during this same period based on immunoblot analysis (data not shown). Measurements made on testes microsomes isolated from the tissues of 3- and 60-d-old animals were within the range of those obtained from d 1–24, but neither 17,20-lyase nor aromatase activities were stimulated by recombinant reductase addition (data not shown).



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  		Figure 4. Testicular weight (g; panel A), total testicular microsomal P450 concentration (nmol/mg; panel B), and activities of 17,20-lyase (panel C) and aromatase (panel D) activities (nmol/mg·2 h) in testes microsomes from control boars on d 1, 7, 14, and 24 of neonatal development. Assay details are described in Materials and Methods. Shown are the means ± SEM and comparisons were made between adjacent means. *, P < 0.05.

 


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  		Figure 5. Testicular microsomal reductase activity levels in control boars on 1, 7, 14, and 24 d of neonatal development. Assay details are described in Materials and Methods. Shown are the means ± SEM. *, P < 0.05. *, P < 0.01; **, P < 0.001.

 
Treatment with the GnRH antagonist had profound effects on testicular development and function. There was no change in plasma LH levels with time in either control or treated piglets and the data were combined. Treatment with the GnRH antagonist successfully suppressed LH concentrations to 50% or less of controls (1.1 ± 0.1 vs. 0.5 ± 0.1 ng/ml in control and treated piglets, respectively; P < 0.01). This was associated with a dramatic inhibition of testicular growth and steroid secretion (Fig. 6Go). The 1.7-fold increase testicular weight that occurred between d 1 and 7 in control piglets was attenuated to just 1.2-fold in treated animals, and the 3.5-fold increase between d 7 and 14 was reduced to an increase of less than 2-fold (Fig. 6AGo). In effect, GnRH antagonist treatment decreased testis weight by 27% on d 7, and 65% on d 14, (P < 0.001). Plasma testosterone concentrations doubled between d 7 and 14 in control piglets, but in GnRH antagonist-treated piglets, were 60% of controls on d 7, and were 35% of controls on d 14 (Fig. 6BGo). There was no significant increase in testosterone concentration in treated pigs between d 7 and 14.



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  		Figure 6. Effect of LH suppression on testicular weight (A) and plasma testosterone concentrations (B) in neonatal boars. The data for control (black bars) and GnRH antagonist (SB75)-treated animals (open bars) for d 7 and 14 groups are expressed as the mean ± SEM. *, P < 0.05.

 
Despite the significant effects of treatment on plasma LH, testicular weight, and testosterone concentrations, the effects of GnRH antagonist treatment on other testicular parameters measured were more variable. There were no day or treatment by day interactions for total P450 concentration, 17,20-lyase or aromatase activities, so data for d 7 and d 14 were combined within treatment and control groups (Table 1Go). Total microsomal P450 concentration was reduced (P < 0.05) by treatment to 64% of controls. The levels of 17,20-lyase activity were significantly reduced also (P < 0.05), but only by 20%. There was a greater effect of GnRH-antagonist treatment on aromatase activity, which was reduced by over 50%. The greater suppression of aromatase compared with 17,20-lyase activity in piglets receiving the GnRH antagonist resulted in an even greater difference between the levels of activity of these two enzymes, such that 17,20-lyase was over 300-fold higher than aromatase on both d 7 and 14. The results of Western immunoblot analyses were consistent with these data (Fig. 7Go). In contrast, to the effects of treatment on testes weight, total P450 concentration and P450 enzyme activities, and regardless of changes in levels with age, there was no effect of GnRH antagonist treatment on reductase activity (Table 1Go) or levels of immunodetectable protein. Similarly, there were no effects of treatment on cytochrome b5 expression (Fig. 7Go).


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  		Table 1. Effects of the GnRH antagonist (SB75) on total cytochrome P450 (total P450), 17,20-lyase, P450arom, and NADPH-cytochrome P450 reductase (reductase) activities of testicular microsomes from neonatal boars

 


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  		Figure 7. Levels of P450c17, cytochrome b5 (Cyt-b5), NADPH-cytochrome P450 reductase (Red), and aromatase cytochrome P450 (Arom) in testicular microsomes from control (C) or treated (T) piglets at d 7 or 14 d of age. Treated piglets received a GnRH antagonist at one d of age, and testes were harvested on d 7 or 14. Enzyme (P450c17 or Arom) and redox partner protein (Cyt-b5 or Red) were detected by immunoblot analysis. Note piglets no. 108 on d 7 and no. 406 on d 14 failed to exhibit a decrease in testes weight, plasma LH or testosterone concentrations. Each lane contains 10 µg of microsomal protein. The arrows indicate the apparent molecular size, and the number under each lane represents individual animals for the 7 d (left panel) and the 14 d (right panel) treatment groups. The standard (+C), run with each gel, represents 10 µg of an archival pig testes microsomal preparation.

 
Finally, it is noteworthy that among the GnRH antagonist treated piglets, there were two animals, one on d 7 (no. 108) and another on d 14 (no. 406), that were clearly different from the others in their respective groups and influenced the means considerably (Table 1Go). It would appear that these animals either failed to receive the entire dose, or represent nonresponders, because all measured parameters fell within the range typical of controls. In fact, these animals helped to establish a clear and strong correlation among LH levels, testicular growth, enzyme activities, and protein expression (Fig. 7Go).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study examined, and directly compared, the activities of P450c17, P450arom, and reductase in porcine testicular microsomes during the first 4 wk of neonatal development. Radiometric assays were used to measure P450 (17,20-lyase and aromatase) activities, allowing for a direct estimate of the rate of the final catalytic steps in androgen and estrogen synthesis in each case. The use of 17OH-pregnenolone as an appropriate substrate for measuring 17,20-lyase activity was predicated on the basis of the predominance of {Delta}5-C19 steroid production by the boar testis (56), the relative lack of 3ß-hydroxysteroid dehydrogenase activity (9, 57), and the fact that 17,20-lyase activity is generally slower than 17{alpha}-hydroxylase activity (36, 57, 58), although the relative levels are also affected by the level of reductase (36) and cytochrome b5 (59). 17,20-lyase activity was unable to be saturated at substrate concentrations approaching solubility limits in aqueous media. Similar observations have been reported for studies on bovine P450c17 (60) and for 3ß-hydroxysteroid dehydrogenase in porcine testis microsomes (61). However, activity was linear with time and protein concentration. Thus, the activities measured provide an accurate comparison between ages and treatments, even if they underestimate the maximal capacity.

Circulating androgen and estrogen concentrations increase in the first few weeks of life and decline rapidly in the second month (5, 6, 7). This appears not to be due to a change in the specific activity of either P450c17 or P450arom. Although there were significant increases in both 17,20-lyase and aromatase activities from neonatal d 1 to 7, this was 2-fold or less. Consistent with previous studies, peripheral testosterone concentrations doubled between 7 and 14 d of age. However, no corresponding major fluctuations were noted in either microsomal 17,20-lyase or aromatase activities in the present study. Levels of 17,20-lyase activity were similar on d 7 and 14 (64.8 ± 1.8 and 66.8 ± 3.9 nmol/mg·2 h, respectively), and there was no significant correlation with testosterone concentration overall. Therefore, the increase in circulating sex steroid seen in the neonatal period is unlikely to be associated with an increase in cellular expression of either P450c17 or P450arom but more probably is due to an overall increase in interstitial volume accompanying an increase in testicular weight of almost 10-fold. This is twice the rate of whole body growth and the proportionate increases in the weights of visceral organs, including liver (62). Treatment markedly suppressed testicular growth and the increase in peripheral testosterone concentrations by half. However, testes weight still doubled in the treated piglets and 17,20-lyase activity was decreased by only 20%. Whether or not there are changes in hepatic clearance of steroids in the neonate as there are during puberty (8, 63) is unknown, but a 4-fold increase in liver weight (62), and presumably blood flow, is likely to be an important determinant of circulating steroid levels. It appears from these data that the specific level of enzyme expression, even if related to total organ size and therefore steroidogenic capacity, cannot be considered the sole determinant of circulating steroid concentrations.

The present study also documents the influence of gonadotropins on the expression of microsomal P450 enzymes, and of the essential redox partner flavoprotein, reductase, and cytochrome b5, during gonadal development. Our data indicate that, in vivo, the expression of P450c17 in the porcine neonatal testis is less sensitive, or less dependent, on gonadotropic support than P450arom. P450c17 expression was inhibited by 20%, whereas P450arom were halved, by GnRH antagonist treatment. LH stimulates aromatase activity (64, 65) and androgen secretion (66, 67) of porcine Leydig cells in vitro, but direct comparisons of enzyme activities or expression have not been reported either in vitro or in vivo. The levels of reductase reported here were high, relative to activities measured in pig liver microsomes (data not shown), though not as high as previous estimates (36). However, we also observed considerable variability with age, as much as a 5-fold difference between d 7 and 24. Moreover, reductase levels changed without any apparent relationship to P450c17, P450arom or total microsomal P450 concentrations, even though few other functions have been ascribed to reductase than the support of microsomal P450 activity (68). Cytochrome b5 is believed to support 17,20-lyase activity of porcine P450c17 (38, 59), and also to stimulate the synthesis of the {Delta}16-steroids by P450c17 (59, 69, 70). Cytochrome b5 expression is unusually high in the porcine testis (42), human (71), and nonhuman primate adrenal zona reticularis (54) and in theca interna of several species (Conley, A. J., and S. M. Mapes, unpublished observations), all tissues with the capacity to synthesize C-19 steroids. The appearance in androgen synthesizing tissues might be equally indicative of regulation of cytochrome b5 expression as it is with the likely involvement of this protein in promoting 17,20-lyase activity. However, as with reductase (72), relatively little is known concerning the regulation of expression of cytochrome b5. Therefore, it is interesting that neither reductase nor cytochrome b5 appears to be affected by gonadotropin support, and that the suppression of androgens themselves had little impact on cytochrome b5 expression.

The results of enzyme activities presented have additional relevance to the control of androgen and estrogen synthesis in the gonads. Despite the possibility that the maximal capacity for 17,20-lyase activity may have been greater, direct comparisons with aromatase activity indicate that the production of C-19 steroids, potential substrates for subsequent aromatization, exceeded the aromatizing capacity of the testes by over 2 orders of magnitude. Differences between androgen and estrogen concentrations in testicular vein approach this level (1) and are consistent with these data. Doubling the available reductase by addition of recombinant protein failed to stimulate either aromatase or 17,20-lyase, so both P450s appear to be saturated with respect to redox protein support. Thus, in contrast to the liver (43), competition between microsomal P450s for limited reductase appears not to be an important factor regulating enzyme activity in the testes. Because the turn-over numbers of porcine P450c17 (59, 73) and P450arom (74) are similar, the differences in activity probably reflect the relative levels of the enzymes themselves, and this is of additional interest in light of total P450 content, as discussed below. Regardless, the degree of the difference between activities may be physiologically critical. Although androgens are metabolized by this route, the relatively low levels of aromatase activity suggest that it is unlikely that conversion to estrogens per se could impact androgen levels based on this limited capacity. In fact, a difference of this magnitude may be adaptive by allowing for the regulation of estrogen synthesis without risk of impacting androgen production concomitantly. This may be particularly important where both enzymes are expressed in the same cell, and are therefore presumably regulated by a single gonadotropic stimulus. It will be of interest to compare these data with similar measurements made in the ovarian follicle, wherein different compartments secrete androgens or estrogen in response to different gonadotropins, and C-19 steroid synthesis occurs more to provide substrate for estrogen production than as a primary source of androgen.

It is of equal interest to note that the inhibition of testes development appeared to be associated with a significant suppression of total microsomal P450 concentration to 50% of control levels. Although this is apparently consistent with the reduction in P450arom expression, it is an unlikely explanation. As noted above, in the case of P450c17 and P450arom, enzyme activities provide an estimate of the actual levels of P450 expression because both P450c17 (59, 73) and P450arom (74) turn over their substrates at very similar rates. Thus, the greatest change noted with gonadotropin suppression was a 50% decrease of P450arom, but of the two enzymes examined it is the one likely to be in lowest abundance. Conversely, the estimated 20% decrease in P450c17 levels is inadequate to bring about the marked reduction in gonadotropin supported, microsomal P450 in porcine testes. No similar studies have been conducted in porcine testes to our knowledge, however, microsomal and mitochondrial P450 spectra have been measured in subcellular fractions isolated from rat and mouse (75, 76). Purvis and colleagues (77, 78) demonstrated that levels of mitochondrial P450 were 40–80 times lower than microsomal P450 levels in rat testes, but were increased many fold in response to human chorionic gonadotropin (hCG) stimulation. Testicular cytochrome b5 levels were changed very little by hCG treatment in comparison to either microsomal or mitochondrial P450 (77), consistent with the observations for the neonatal pig reported here. Unlike the rat, mouse testicular mitochondrial and microsomal P450 concentrations were quite similar and both declined equally following a desensitizing dose of hCG (75, 76). Although decreases in specific enzyme activities were shown to accompany the decline in P450, no attempt was made to determine the relative contribution of each to the total P450 concentration.

However, the apparent decrease in microsomal P450 in response to GnRH antagonist treatment must be interpreted with caution. Both Purvis et al. (78) and Luketich et al. (76) observed high levels of cytochrome oxidase in mitochondria that generated a trough from 430–435 nm and potentially complicated P450 determinations in their fractions. A similar trough was observed in our samples. As suggested by Purvis et al. (78), we recalculated our data, according to the method of Kowal et al. (51), to minimize any potential confounding effects of cytochrome oxidase on P450 determinations, but our reported estimates did not change significantly nor did it alter the treatment differences we noted. Whether or not the trough seen at 430–435 nm represents cytochrome oxidase or other mitochondrial pigments, the P450 concentrations we report could be underestimated. Nonetheless, we believe that any difference is small for two reasons. Firstly, we demonstrated that the degree of mitochondrial contamination in our microsomal preparations was less than 10% of the protein fraction (Fig. 1Go). Secondly, our estimates are not only very similar to those reported for porcine testes microsomes by Mason et al. (42), they are slightly higher in fact. The apparent decrease in microsomal P450 could have been biased by, or reflect changes actually occurring in, mitochondria even at low levels of contamination if mitochondrial P450 levels were high. This also seems unlikely. Mitochondrial P450 was reported to be lower (if only modestly) than microsomal P450 concentrations in the testes of the pig (42), rat (77, 78), and mouse (76). If the apparent the decline in microsomal P450 concentration following GnRH antagonist treatment in neonatal pig testes is real, but cannot be accounted for by either P450c17 or P450arom, what P450(s) does it represent? Cytochrome P450 1A2 (79, 80), and other microsomal P450s (81) have been identified in rat testes, and 2{alpha}-, 6ß-, and 16-hydroxylase activities have been observed in testicular microsomes from bulls (82), but no candidate microsomal P450s can be suggested at this time that are likely to be expressed in abundance in the gonads of the pig. The high level of reductase observed here suggests that any would be well supported. What is most intriguing is that, whatever their identity, their expression appears to be sensitive to, and dependent on, continued gonadotropic stimulation.

In summary, we have shown that the perinatal androgen and estrogen rise in the male pig is unlikely to result from an increase in the testicular expression of either P450c17 or P450arom, or from changes in redox protein (reductase and cytochrome b5) support, but more probably from an increase in testicular interstitial mass. In addition, the levels of testicular P450c17 were orders of magnitude higher than those of P450arom. Consequently, the level of aromatase activity is not predicted to be enough to metabolize sufficient substrate to impact gonadal androgen secretion. Inhibition of gonadotropic support markedly suppressed neonatal testes growth, and inhibited the expression of total microsomal P450, including both P450c17 and P450arom, but P450arom appeared to be more sensitive to gonadotropic stimulation than did P450c17. In addition, it appeared that these two enzymes could not account for the total pool of microsomal P450 in the neonatal testes, despite it being clearly gonadotropin sensitive. It will be of interest to determine the identity of these presumably nonsteroidogenic, microsomal P450s in future studies. Regardless of this large, total P450 pool, reductase appears to be saturating for both P450c17 and P45arom. Furthermore, the expression of both reductase and cytochrome b5 appears to be constitutive, and not reliant on gonadotropic support.


   Acknowledgments
 
The authors wish to thank Dr. T. Wise for assistance with steroid RIAs, and the Meat Animal Research Center swine unit for care of the animals.


   Footnotes
 
Abbreviations: hCG, Human chorionic gonadotropin; NADPH, nicotinamide adenine dinucleotide phosphate, reduced form.

Received March 20, 2002.

Accepted for publication May 6, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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