Adrenal function in Angora goats: A comparative study of adrenal steroidogenesis in Angora goats, Boer goats, and Merino sheep1 Y. Engelbrecht and P. Swart2 University of Stellenbosch, Stellenbosch, South Africa, 7602

ABSTRACT: South African Angora goats (Capra aegagrus) are susceptible to stress conditions, possibly due to adrenal cortex malfunction. Selection for mohair production may reduce adrenal function and decrease cortisol production. Secretion of cortisol by the adrenal cortex is essential for the induction of several gluconeogenic enzymes that enable animals to survive stressful conditions, and adrenocortical insufficiency, therefore, precipitates a vulnerability to stress. In this study, Angora goats were compared with two breeds generally accepted as hardy, Boer goats (Capra hircus) and Merino sheep (Ovis aries). Adrenal steroidogenesis was studied using subcellular fractions prepared from the

adrenal glands of freshly slaughtered animals. Adrenal microsomes and mitochondria were incubated with the relevant steroid substrates, and products were analyzed and quantified with TLC, HPLC, or RIA. Subsequently, the activity of individual enzymes involved in this pathway were further investigated. The cytochrome P450 content in the preparations was also compared. The results from these studies indicated that the activity of the cytochrome P450c17 enzyme in Angora goats differed (P < .01) from that of the other species investigated. This difference may contribute to the cause of the observed hypoadrenocorticism in Angora goats.

Key Words: Angora, Goats, Hydrocortisone, Mohair, Sheep, Stress 2000 American Society of Animal Science. All rights reserved.

Introduction Angora goats (Capra aegagrus) are an important fiber-producing small stock breed. South Africa produces approximately 40% of the global mohair crop, and locally the Angora industry plays an important role in employment opportunities. Unfortunately, this high fiber production is negatively related to fitness traits (Snyman and Olivier, 1996), and severe losses of Angora goats during cold spells present a serious problem to mohair producers. Previous research showed that an abrupt drop in blood glucose concentrations was the crucial factor responsible for the inability to produce metabolic heat (Wentzel et al., 1979; Wentzel, 1987). The susceptibility of Angora goats to minor

1 This study was supported by the FRD and the University of Stellenbosch. 2 The authors would like to thank Robert Tuckey, University of Western Australia, for his assistance with the mitochondrial assays and his kind donation of pregnenolone antibodies and cyanoketone for these assays. We also would like to thank J. I. Mason for donating the 3βHSD antiserum. Finally, we thank Thielmann Niewhoudt and Werner Ferreira for kindly donating animals for this research. 3 Correspondence: Department of Biochemistry, University of Stellenbosch, Private Bag X1, Matieland, 7602, South Africa (fax: 27218083022; E-mail: [email protected]). Received February 11, 1999. Accepted August 21, 1999.

J. Anim. Sci. 2000. 78:1036–1046

stress conditions could be attributed to a condition of hypoadrenocorticism, because Van Rensburg (1973) reported that the selection for mohair production reduced adrenal function with a subsequent decrease in cortisol production. In addition, the stimulation of the Angora hypothalamic-pituitary-adrenal axis with insulin and ACTH in vivo resulted in less cortisol production by Angora goats than by Boer goats and Merino sheep (Engelbrecht et al., 1999). Adrenal steroid biosynthesis is a complex process that takes place in two organelles: mitochondria and smooth endoplasmic reticulum (ER). Cholesterol is the common precursor, and metabolic intermediates move back and forth between these two subcellular compartments, where different types of cytochrome P450 (P450) are involved (Takemori and Kominami, 1984). In this study, adrenal steroidogenesis was investigated as a possible cause of the lowered cortisol production in Angora goats. The activity of the enzymes involved in adrenal microsomal and mitochondrial steroidogenesis in Angora goats were compared with that of two breeds generally accepted as hardy, Boer goats (Capra hircus) and Merino sheep (Ovis aries).

Materials and Methods Animals For the microsomal experiments, eight Angora goat ewes, eight Boer goat ewes, and seven Merino sheep

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ewes were slaughtered for collection of their adrenal glands. These animals were bred and kept at the Grootfontein Agricultural Institute at Middelburg in the Northern Cape, South Africa. They were 6 mo of age and given ad libitum access to ground alfalfa hay. For the mitochondrial preparations, material was collected either from the local abattoir (Merino sheep) or from local farmers (Angora and Boer goats). The animals were between 2 and 6 yr old and from both sexes. In the selection for Angora goat material, care was taken to use animals that produced hair of high quality and quantity.

Preparation of Microsomes Adrenal microsomes were prepared as previously described (Swart et al., 1995, 1996). Animals were slaughtered, and the adrenals were immediately removed and stored at −20°C. All subsequent procedures were performed at 4°C. After removal of the capsule, the adrenals were homogenized with four to five volumes of a .25 M sucrose solution (pH 7.4). The adrenal homogenates were centrifuged at 850 × g for 8 min. The cell debris was discarded, and the supernatants were centrifuged at 21,500 × g for 10 min. The 21,500 × g supernatant fraction was centrifuged for an additional 60 min at 70,000 × g to obtain a microsomal pellet. This pellet was resuspended in a .15 M KCl solution and centrifuged for 30 min at 70,000 × g. The resulting pellet was resuspended in 100 mM potassium phosphate buffer (pH 7.2) that contained 20% glycerol, 100 ␮mM EDTA, and 100 mM dithiothreitol. The microsomal subcellular fractions were pooled within species; thus, species and preparation were confounded.

Preparation of Mitochondria Adrenal mitochondria were prepared according to previously published methods (Cheng and Harding, 1973; Swart et al., 1988). Animals were slaughtered, and the adrenals were kept on ice. After removal of the capsule, the adrenals were homogenized with four volumes of a .25 M sucrose solution that contained 1 mM EDTA (pH 7.4). The homogenate was centrifuged at 650 × g for 10 min and the supernatant subsequently at 6,700 × g for 15 min. The resulting pellet was washed with a .25 M sucrose solution that contained 1 mM EDTA and 1% BSA (pH 7.4). After centrifugation at 10,400 × g for 15 min, the mitochondrial pellet was resuspended in the .25 M sucrose solution without BSA (pH 7.4). The mitochondria were pooled within species; thus, species, preparation, and slaughter procedures were confounded. The mitochondrial suspension was stored at 4°C, and all metabolic experiments were completed within 6 h after preparation.

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1995). Incubations were performed in 1.5-mL Eppindorf tubes in a shaking waterbath at 37°C in a total volume of 400 ␮L. The reaction was carried out in a 50 mM Tris buffer solution (pH 7.4) that contained 1% BSA and 50 mM NaCl. In addition, the reaction mixture contained 10 ␮M progesterone, 17OH-progesterone, or pregnenolone (Sigma Chem Co., St. Louis, MO), .44 ␮mol MgCl2, and 398 mU of isocitrate dehydrogenase (Sigma Chemical Co.). After a 10-min preincubation period, the appropriate adrenal microsome preparation was added, and the system was incubated for an additional 5 min. The reaction was initiated by the addition of 420 nmol isocitrate (Merck, Darmstadt, Germany) and .44 ␮mol NADPH (Boehringer, Mannheim, Germany). When the activity of individual enzymes was studied, the corresponding cofactor was used (Fevold et al., 1978). At regular time intervals, 50 ␮L of reaction mixture was removed and thoroughly mixed with a cold mixture of 5 mL of methylene chloride and 450 ␮L of H2O. Extraction of Steroid Metabolites. The steroid metabolites were extracted by separating the organic and water phases using a bench centrifuge at medium speed. The water phase was subsequently aspirated, and the residual methylene chloride extracts were dried under nitrogen. The steroid products were redisolved, either in 100 ␮L of methylene chloride for TLC analysis or 50 ␮L of methanol for HPLC analysis. Chromatography. Thin layer chromatography was carried out on aluminium TLC plates covered with silica gel 60 F254 (Merck). The solvent system used for development was chloroform:ethyl acetate 4:1 (vol/ vol). Steroid standards were cochromatographed to identify the reaction products under ultraviolet light. The individual steroid-containing spots were cut out, and the radioactivity was measured in 8 mL of scintillation fluid (Insta-gel II, Packard Instrument Co., Meriden, CT). The radioactivity of each product was expressed as a percentage of the total radioactivity as determined for a specific sample. For HPLC analysis, the products obtained from the incubation experiments were extracted as described and separated with a Waters 840 liquid chromatograph equipped with a Novapak C18 column at a flow rate of 1 mL/min. The mobile phase consisted of solvent A (water:methanol, 40:60) and solvent B (methanol). Separation was achieved by elution with solvent A for 15 min followed by a linear gradient from 100% A to 100% B for 10min and a further 10 min isocratic elution with solvent B. Tritiated steroids were detected with a Radiomatic A-100 flow detector (Packard Instrument Co.). Steroid concentrations were determined using the integrating facilities of the radioactivity detector. Steroid recovery was greater than 85%.

Microsomal Steroid Metabolism

Mitochondrial Steroid Metabolism

Adrenal microsomal steroid metabolism was studied using techniques previously published (Swart et al.,

Measurement of Cytochrome P450c11 Activity. The activity of P450c11 was determined using the same

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incubation procedure as described for the microsomal preparations. The microsomes were replaced with mitochondria (500 to 1,000 ␮g of mitochondrial protein). Deoxycorticosterone was used as substrate because it was more readily available, and the P450c11 Km values for deoxycortisol and corticosterone are virtually identical. Measurement of Cytochrome P450scc Activity. Incubations were performed at 37°C in a shaking waterbath in glass test tubes (12 × 100 mm) (Tuckey and Cameron, 1993). The incubation buffer contained .25 M sucrose solution, 100 mM HEPES, 40 mM KCl, 10 mM MgSO4, .4 mM EDTA, .012 mM cyanoketone, and .15% fatty acid-free BSA (pH 7.4). To .65 mL of this buffer, 5 mM mitochondrial P450 was added. The reaction was initiated by the addition of 5 mM isocitrate and .5 mM NADPH. The total incubation mixture was .95 mL. Aliquots (25 ␮L) were removed from the incubation mixture at specific time intervals and added to 1 mL of ice-cold ethanol. The pregnenolone content of the samples was subsequently determined with a RIA as previously published (Lambeth et al., 1980; Tuckey and Cameron, 1993). An aliquot of the ethanol solution was dried under N2, and .5 mL of a 100 mM sodium phosphate (pH 7.2), 155 mM NaCl, .1% (wt/vol) gelatin, .1% (wt/vol) Na-azide solution containing 260 Bq [7(n)3 H]pregnenolone (.3 pmol) was added to the residue. Antiserum against pregnenolone (200 ␮L, diluted 1:5,600) was added, and the samples were incubated at 4°C for 16 h. Bound and free steroids were separated by addition of 200 ␮L of dextran-coated charcoal (6.25 g/L activated charcoal and .625 g/L dextran T-70 in 100 mM sodium phosphate as previously described). The samples were subsequently incubated for 20 min at 4°C followed by centrifugation at 3,000 × g for 30 min. The supernatant containing the unbound pregnenolone was removed and added to 4 mL of scintillation fluid (Insta-gel II), and the radioactivty was determined. Pregnenolone standards (.02 to 2 ng) were analyzed simultaneously.

Electrophoresis and Immunoblot Analysis An SDS-PAGE procedure was performed as previously described (Weber and Osborn, 1969). Western blot analysis was performed according to the method of Strott (1989).

Protein Determinations Protein concentrations were determined using the bicinchoninic acid method (Pierce and Suelter, 1977; Smith et al., 1985) (Pierce, Rockford, IL) with BSA (2 mg/mL) as a protein standard, according to the manufacturer’s instructions.

Assay of Cytochrome P450 The microsomal and mitochondrial samples were saturated with CO for 60 s. After a baseline was re-

corded between 400 and 500 nm, a few grains of dithionite were dissolved in the sample cuvette by gentle inversion. The resulting difference spectrum was recorded between 400 and 500 nm, and the cytochrome P450 concentration was calculated using an extinction coefficient of 92 cm−1ⴢmM−1 and the difference in absorbance between 450 and 490 nm (Omura and Sato, 1964).

Statistical Analysis The results are presented as mean ± SE and represent pools of microsomes or mitochondria from at least three experimental animals. The data were evaluated with GraphPad Software Version 2 (San Diego, CA). In Figures 2 and 4, the model was time (Dunnett’s test), breed (Bonferroni’s test), and residual. In Figure 5, it was breed (Bonferroni’s test) and residual. A Pvalue less than .05 was considered significant. Species and preparation were confounded for microsomes, and species, preparation, and slaughter procedures were confounded for mitochondria. However, because of the number of animals for each species represented in each preparation and the repeatability of the procedures, species was most likely the major source of differences when they occurred in response to treatments.

RESULTS Cytochrome P450 Content The adrenal microsomal P450 content in Angora goats, Boer goats, and Merino sheep were .37, .39, and .33 mmol/mg of protein, respectively, and the adrenal mitochondrial P450 content for the same animals was .35, .44, and 1.36 pmol/mg of protein, respectively.

Activity of Microsomal Steroidogenic Enzymes The metabolism of pregnenolone, to determine the ratio of the production of glucocorticosteroid precursors (deoxycorticosterone and deoxycortisol) to that of dehydroepiandrosterone (DHEA) and(or) androstenedione, was studied. The metabolites obtained from pregnenolone were separated with HPLC and are listed in Table 1. In a 10-min incubation period, Angora goat microsomes produced less (P < .05) glucocorticosteroid precursors than Boer goat and Merino sheep microsomes. The Angora adrenal microsomes converted 36% of the pregnenolone in the incubation mixture to the glucocorticoid precursors, deoxycortisol and deoxycorticosterone, and the other two species converted 79 and 82%, respectively. In contrast, Angora goat produced more (P < .05, n = 4) 17OH-pregnenolone and DHEA than did the other two species. Progesterone was subsequently incubated with Angora goat, Boer goat, and Merino sheep adrenal microsomes. The metabolites were extracted at specific time intervals, separated, and analyzed with HPLC (Figure

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Table 1. Metabolite distribution (mean ± SE, n = 4) of 10 ␮M pregnenolone metabolism in Angora goat, Boer goat, and Merino sheep adrenal microsomes (.32 mM P450). Incubation mixtures contained NADPH and NAD+. The data were evaluated with one-way ANOVA, followed by Bonferroni’s multiple comparison test

Animal Angora goat Boer goat Merino

Percentage of glucocorticosteroid precursors formed (DOC + DOCL)

Percentage of DHEA and 17-OH pregnenolone formeda

Percentage of androstenedione formed

Percentage of pregnenolone remaining

35.6b ± 8.9 78.5c ± 13.9 82.03c ± 6.5

34.7d ± 4.2 8.8e ± 1.7 0e ± 0

5.5 ± 2.33 — —

1.06 ± 1.06 6.3 ± 4 —

DOC: Deoxycorticosterone, DOCL: Deoxycortisol, DHEA: Dehydroepiandrostenedione. The steroid intermediates, progesterone and 17-OH progesterone, are not indicated. a Efficient separation and quantification was not always possible. b,c Within columns, values without common superscripts differ (P < .05). d,e Within columns, values without common superscripts differ (P < .001).

1). A time course for the formation of the steroid products is shown in Figure 2. In all three species, progesterone was completely metabolized after 15 min. The initial reaction rates for progesterone metabolism are given in Table 2. After 15 min (Figure 2), 2.82 ± .024, 2.58 ± .048, and 2.18 ± .068 nmol of deoxycorticosterone and 1.016 ± .024, 1.212 ± .056, and 1.448 ± .024 nmol of deoxycortisol were produced by Angora goat, Boer goat, and Merino sheep adrenal microsomes, respectively. At this time, Angora goat produced more (P < .001) deoxycorticosterone and less (P < .01) deoxycortisol than the other species (Figure 2). Androstenedione and 17OH-progesterone production was less than 5% in all three species. The activity of specific enzymes in the adrenal microsomal steroidogenic pathway were subsequently studied. Pregnenolone metabolism could be manipulated by the selective addition of cofactors (Figure 3). The activity of 3β-hydroxysteroid dehydrogenase/∆5-∆4 isomerase (3βHSD) was investigated by measuring the conversion of pregnenolone to progesterone using NAD+ as cofactor in the absence of NADPH. Results obtained from these experiments are summarized in Figure 4a (see also Table 2 and Figure 3c). Addition of NAD+ resulted in the formation of progesterone in all three species. As indicated in Figure 4a, there was no difference (P > .05) in the activity of 3βHSD among species (see table at bottom of figure). The activity of cytochrome P450 17α-hydroxylase/17-20 lyase (P450c17) for pregnenolone conversion to DHEA was studied using NADPH as cofactor in the absence of NAD+ (Figure 4b, see also Figure 3b and Table 2). In Angora goat adrenal microsomes, P450c17 converted pregnenolone to DHEA faster (P < .01) than in the other two species. Cytochrome P450c21 (P450c21) activity was investigated by incubating the adrenal microsomes with 17OH-progesterone. The results are presented in Figure 5. In all three species, more than 90% ± .9 of the 17OH-progesterone was converted to deoxycorticosterone, with very little conversion to androstenedione. There was no difference (P > .05) in the activity of P450c21 among species.

Activity of Mitochondrial Steroidogenic Enzymes The activity of P450c11 was investigated using the conversion of deoxycorticosterone to corticosterone. The results are summarized in Table 3. The initial reaction rate of Boer goat P450c11 was faster (P < .05) than the other experimental animals. The P450scc activity was determined by the conversion of 22ROHcholesterol to pregnenolone (Table 3). The initial reaction rate of Boer goat enzyme was faster (P < .05) than that of Merino sheep, with no difference in the activities of Angora and Boer goat, and Angora goat and Merino sheep, respectively.

SDS-PAGE and Western Blot Analysis Angora goat, Boer goat, and Merino sheep adrenal microsomes were subjected to SDS-PAGE and subsequent Western blot analysis with rabbit anti-sheep adrenal P450c21 and rabbit anti-human placental 3βHSD serum (Figure 6). No obvious differences could be detected in the three SDS-PAGE profiles (Figure 6a). Western blot analysis with anti-sheep adrenal P450c21 showed a single band corresponding with a molecular mass of 55,000 Da for all three species, which is in good accordance with results previously published for P450c21 (Figure 6b) (Kominami et al., 1980). Similar results were obtained from Western blot analysis of microsomes using rabbit anti-human placental 3βHSD serum. The 3βHSD showed as a single band corresponding to a molecular mass of 40,000 Da (Figure 6c) (Ford and Engel, 1974).

Discussion In adrenal mitochondria, two P450-dependent enzymes, P450scc and P450c11, are responsible for the first and terminal steps in adrenal glucocorticoid biosynthesis (Figure 7) (Takemori and Kominami, 1984). Pregnenolone, produced from cholesterol by the action of P450scc, migrates from the mitochondria to the endoplasmic reticulum. The subsequent pregnenolone

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Figure 1. Typical HPLC elution profiles of 10 ␮M Angora goat progesterone metabolites separated on a C18 column (280 × 6 mm) at a) 0, b) 6, and c) 15 min. The mobile phase consisted of solvent A (water:methanol, 40:60) and solvent B (methanol). Separation was achieved by elution with solvent A for 15 min followed by a linear gradient from 100% A to 100% B for 10 min and a further 10-min isocratic elution with solvent B at a flow rate of 2 mL/ min. Steroid concentrations were determined from the peak areas (a linear relation was found between concentration and peak area) (PROG: progesterone, DOC: deoxycorticosterone, DOCL: deoxycortisol).

Figure 2. Time course of progesterone (PROG) metabolism by Angora goat (A), Boer goat (B), and Merino sheep (M) adrenal microsomes. The microsomes (.32 mM P450) were incubated with 10 ␮M progesterone at 37°C with constant stirring. The products were extracted and subsequently separated and quantified with a HPLC system as described elsewhere. The results were statistically analyzed by comparing the species’ cortisol levels to that at 0 min and are indicated on the respective graphs (repeated measures ANOVA, followed by Dunnet). The cortisol levels of each species were also compared with the response of the other animals (repeated measures ANOVA, followed by Bonferroni) (inset: Statistical comparison). Androstenedione and 17OH-progesterone production were insignificant (DOC: deoxycorticosterone, DOCL: deoxycortisol).

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Table 2. Initial reaction rates (mean ± SE, n = 4) for 10 ␮M progesterone metabolism and 3βHSD and P450c17 activity by Angora goat, Boer goat, and Merino sheep adrenal microsomes. The data were evaluated with one-way ANOVA, followed by Bonferroni’s multiple comparison test Progesterone metabolism

Animal Angora goat Boer goat Merino sheep

Progesterone utilizationa

DOC produceda

Deoxycortisol produceda

3βHSD activitya Progesterone produced

.42d ± .024 .71e ± .035 .4d ± .036

.3bc ± .008 .35b ± .025 .25c ± .003

.05b ± .03 .15c ± .008 .13bc ± .01

.5 ± .051 .65 ± .028 .53 ± .082

P450c17 activitya DHEA produced 1.13a ± .194 .38b ± .062 .5ab ± .165

3βHSD: 3β-hydroxysteroid dehydrogenas/∆5-∆4 isomerase, DOC: deoxycorticosterone, DHEA: dehydroepiandrosterone. a nmolⴢmL−1ⴢmin−1. b,c Within columns, values without common superscripts differ (P < .05). d,e Within columns, values without common superscripts differ (P < .01).

metabolism is the cornerstone of adrenal cortical function in mammals (Conley and Bird, 1997). It is therefore believed that one particularly critical regulatory juncture involves the differential metabolism of pregnenolone, which can be utilized by two enzymes in competing reactions (Shinzawa et al., 1985; Conley and Bird, 1997). Cytochrome P450c17 catalyzes the biosynthesis of DHEA from pregnenolone, which otherwise can be metabolized to progesterone by the enzyme 3βHSD and subsequently the glucocorticoids, by the actions of P450c17 and steroid 21-hydroxylase, P450c21. Steroid production is regulated largely by the relative levels and tissue-specific array of steroidogenic enzymes expressed at the cellular level (Conley and Bird, 1997). The terminal reaction(s) in glucocorticoid production take place in the adrenal mitochondria, where deoxycorticosterone and deoxycortisol are converted

to corticosterone and cortisol by steroid 11β-hydroxylase, P450c11. Steroidogenesis in the adrenal ER can be divided into two distinct pathways. After the removal of the side chain of cholesterol by P450scc, the resultant ∆5 steroid, pregnenolone, enters the ER from the mitochondria (Takemori and Kominami, 1984). In the ∆5 pathway, pregnenolone is converted to 17OH-pregnenolone and DHEA by steroid 17α-hydroxylase, P450c17. The DHEA is converted to DHEA-sulfate and serves as source for adrenal androgens. The second, or ∆4, pathway, is initiated by the action of the 3βHSD, which catalyses the conversion of the ∆5 steroids to the corresponding ∆4 steroids, progesterone, 17OH-progesterone, and androstenedione (Sasano et al., 1989; Suzuki et al., 1992; Miller et al., 1997). Progesterone, physiologically the most important adrenal steroid intermediate, serves as substrate for the steroid 21-hy-

Figure 3. Typical HPLC elution profiles of 10 ␮M pregnenolone metabolism by Merino adrenal microsomes on a C18 column (150 × 3.9 mm) after 10 min. The mobile phase consisted of solvent A (water:methanol, 40:60) and solvent B (methanol). Separation was achieved by elution with solvent A for 15 min followed by a linear gradient from 100% A to 100% B for 10 min and a further 10-min isocratic elution with solvent B. The flow rate was 1 mL/min. a) Both NAD+ and NADPH were added. b) NADPH alone was added. c) NAD+ alone was added (DOC: deoxycorticosterone, AND: androstenedione, DOCL: deoxycortisol, 17OH-PROG: 17OH-progesterone, 17OH-PREG: 17OH-pregnenolone, DHEA: dehydroepiandrosterone, PROG: progesterone, PREG: pregnenolone).

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Figure 4. a) Comparison of the activity of 3βHSD in Angora goat (A), Boer goat (B), and Merino sheep (M) adrenal microsomes. The activity was compared by measuring the conversion of 10 ␮M pregnenolone to progesterone with only NAD+ as cofactor, and no difference was observed. b) Comparison of the activity of P450c17 as measured by the conversion of 10 ␮M pregnenolone to 17OH-pregnenolone and dehydroepiandrosterone with only NADPH as cofactor. The results were statistically analyzed by comparing the species’ cortisol levels to that at 0 min and are indicated on the respective graphs (repeated measures ANOVA, followed by Dunnet). The cortisol levels of each species were also compared with the response of the other animals (repeated measures ANOVA, followed by Bonferroni) (inset: statistical analyses). The Angora’s P450c17 activity toward pregnenolone was significantly faster than the other two species (3βHSD: 3β-hydroxysteroid dehydrogenase/∆5-∆4 isomerase, P450c17: cytochrome P450c17, PROG: progesterone, DHEA:dehydroepiandrosterone).

droxylase, P450c21, as well as for P450c17. The 21hydroxylation of progesterone yields deoxycorticosterone, the precursor for the mineralocorticoids in mammals, and 17-hydroxylation followed by 21-hydroxylation will yield the glucocorticoid precursor, deoxycortisol (Takemori and Kominami, 1984). In certain animals, such as pigs, 17OH-progesterone may undergo a lyase reaction (removal of the acetyl side chain at position 17) to form androstenedione (Sasano et al., 1989). From the preceding discussion, it is apparent that the three enzymes mentioned, 3βHSD, P450c17, and P450c21, play a pivotal role in channeling metabolites during adrenal steroidogenesis. If a 3βHSD deficiency is encountered, the ∆5 pathway will be favored and adrenal androgen production will be high. In the ∆4 pathway, the relative activities of P450c21 and P450c17 will determine the distribution of the common substrate, progesterone, between mineralocorticoid and glucocorticoid precursors (Hiwatashi and Ichikawa, 1981; Kater and Biglieri, 1994; Conley and Bird, 1997). A P450c21 deficiency will prevent the production of all corticosteroids, whereas a P450c17 deficiency will result in the loss of cortisol production

alone. Many cases of P450c17 deficiencies have been reported in which the activities of 17α-hydroxylase and 17,20-lyase were absent (Monno et al., 1994, 1997; Suzuki et al., 1998). The consequent defects in the synthesis of cortisol and sex steroids cause sexual infantilism and a female phenotype in both genetic sexes, as well as mineralocorticoid excess and hypertension. The existence of true isolated 17,20-lyase deficiency has, however, only recently been confirmed in two patients homozygous for substitution mutations in CYP17, the gene encoding P450c17 (Geller et al., 1997). The progesterone 17-lyase activity exhibited by P450c17 in certain mammals will increase adrenal androgen production during reduced, or in the absence of, P450c21-activity. No detailed study of adrenal steroidogenic activity in Angora goats has been previously published. Because reduced adrenal function is believed to play a significant role in the observed hypoadrenocorticism in Angora goats, it was decided to study adrenal steroidogenesis in this animal to identify the molecular basis of this physiological defect (Van Rensburg, 1973). Identification of differences in the steroidogenic activities of adrenal mitochondria and ER, between Angora

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Figure 5. The products of 10␮M 17OH-progesterone metabolism by Angora goat, Boer goat, and Merino sheep adrenal microsomes. The microsomes were incubated with 10 ␮M 17-OH progesterone for 15 min. Thin layer chromatography was carried out on aluminium silica gel 60 F254 TLC plates. The solvent system used was chloroform:ethyl acetate 4:1 (vol/vol). The radioactivity of each product was expressed as a percentage of the total radioactivity as determined for a specific sample. The data were evaluated with one-way ANOVA, followed by Bonferroni’s multiple comparison test, and no difference in the activity of P450c21 between the species could be detected.

goats and the other two species, should provide insights into the possible causes of hypoadrenocorticism in Angora goats. The current investigation of the mitochondrial P450scc and P450c11 activities in Angora goats, Boer goats, and Merino sheep revealed no significant differences that might contribute to the impaired glucocorticoid production in Angora goats. Significant differences were, however, observed in the metabolism of pregnenolone and progesterone by the adrenal microsomes of the three species. When pregnenolone was used as substrate, in the presence of both NAD+ (re-

quired for 3βHSD-activity) and NADPH (required for P450-dependent steroid hydoxylase activity), Angora goat produced approximately 40% less glucocorticoid precursors than Boer goat and Merino sheep, respectively. This finding indicated a possible augmented activity of Angora P450c17. This enzyme exhibited a greater affinity for the ∆5-steroid pathway and produced significantly more ∆5 products than ∆4 products, which are essential precursors for the production of corticosteroids. This result indicated a preference of Angora adrenal for the conversion of pregnenolone to 17OH-pregnenolone and ultimately DHEA. The flow of metabolites through the adrenal microsomal steroidogenic pathway could be effectively controlled by the two cofactors NADPH and NAD+. When NADPH alone was supplied with pregnenolone as substrate, Angora goat adrenal microsomes were the most efficient in converting the ∆5-steroid to DHEA, and approximately 40% more of the total pregnenolone added was converted to DHEA than in the other two species. This result clearly indicated that, as far as pregnenolone was concerned, Angora P450c17 was more active than the other two species. This activity of the Angora P450c17 will certainly have an effect on cortisol production in the animal. In the presence of only NAD+, all three species converted pregnenolone to progesterone at an equal rate, and it can therefore be assumed that the 3βHSD-activity will not be the cause of the Angora’s inability to produce sufficient cortisol. It is interesting to note that the conversion of DHEA to androstenedione by 3βHSD was extremely low in all the adrenal preparations investigated (Angora goat, Boer goat, and Merino sheep) independent of the method of steroid analysis (HPLC or TLC). A comparison of progesterone metabolism by Angora goat, Boer goat, and Merino sheep adrenal microsomes confirmed that Angora goat did not have the same capacity for glucocorticoid production than the other experimental animals. Although deoxycorticosterone production was essentially identical in all three species, indicating no difference in P450c21 activities, the deoxycortisol production differed markedly. This indicates that Angora P450c17 has a lower affinity for progesterone as substrate than the other species. The

Table 3. Initial reaction rates (mean ± SE, n = 4) for 10 ␮M deoxycorticosterone metabolism and the conversion of 50 ␮M 22ROH-cholesterol to pregnenolone by Angora goat, Boer goat, and Merino sheep adrenal mitochondria. The data were evaluated with one-way ANOVA, followed by Bonferroni’s multiple comparison test Animal Angora goat Boer goat Merino sheep

Cytochrome 11β activitya (corticosteroid production)

Cytochrome P450scc activitya (pregnenolone production)

1.64 ± .46 9.43b ± 1.14 1.86a ± .24

238 ± 63.9 343 ± 131 153 ± 47.8

pmolⴢmg−1 proteinⴢmin−1. Within columns values without common superscripts differ (P < .001).

a

b,c

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Figure 6. a) SDS-PAGE gel (12% T, 2.7% T) of Angora goat (lanes 2 and 3), Boer goat (lanes 4 and 5), and Merino sheep (lane 7) adrenal microsomes. The proteins were visualized by staining with Coomassie brilliant blue. Lanes 1 and 8: molecular marker. b) Immunoblot of Angora goat (lanes 2 and 3), Boer goat (lanes 4 and 5), and Merino sheep adrenal microsomes (lanes 6 and 7); detected with sheep P450c21 antiserum (1:5,000 dilution). Lanes 1 and 8: molecular marker. Lanes 2, 4, and 6: 5 mg protein. Lanes 3, 5, and 7; 10 mg protein. c) Immunoblot of Angora goat (lanes 2 and 6), Boer goat (lanes 3 and 7), and Merino sheep (lanes 4 and 8) adrenal microsomes (5 mg protein); detected with 3βHSD antiserum) lanes 2, 3, and 4: 1:500 dilution; lanes 6, 7, and 8: 1:1,000 dilution). Lanes 1 and 5: molecular marker. Electrophoresis and immunoblotting were carried out as described in the text, with migration from top to bottom.

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Figure 7. Schematic representation of adrenal steroidogenesis. CHOL: cholesterol, PREG: pregnenolone, 17OHPREG: 17OH-pregnenolone, DHEA: dehydroepiandrosterone, PROG: progesterone, 17OH-PROG: 17OH-progesterone, AND: androstenedione, DOC: deoxycorticosterone, DOCL: deoxycortisol, CORT: cortisol. conversion of 17OH-progesterone to deoxycortisol was also identical in all three species and confirmed that Angora P450c21 was indeed as active and had the same substrate specificity as in the other two animals. The results obtained in this study indicate a difference in the activity of P450c17 between Angora goats and the two more hardy species, Boer goats and Merino sheep. These two animals consequently have a higher potential for glucocorticoid production than Angoras. It was not possible, using the crude subcellular preparations, to determine whether the Angora’s preference for the ∆5 pathway would eventually lead to higher adrenal androgen production in vivo. From the results obtained in this study, it seems likely that this could be the case because no significant lyase activity toward the ∆4 steroids was detected. The regulation of energy metabolism to cope with adverse environmental conditions is a complex process in all mammals; therefore, the difference in glucocorticoid production between Angora goats and the other two experimental animals, used in this study, cannot be taken as the only cause of hypoadrenocorticism in Angora. The apparent preference by Angora P450c17 for the ∆5-pathway will, however, certainly contribute to the inability of the animal to raise its cortisol levels sufficiently under cold stress. This first investigation of Angora adrenal steroidogenesis has now opened the way for further experiments, which will concentrate on adrenal P450c17-activty.

Implications The study of adrenal steroidogenesis yielded a difference in glucocorticoid production between Angora

goats, Boer goats, and Merino sheep. The apparent preference exhibited by Angora P450c17 for the ∆5pathway during adrenal steroidogenesis will contribute to a lowered production of glucocorticoids. This phenomenon, however, will probably be chronic in vivo and have a long-term effect on the carbohydrate metabolism of Angora goats. The critical balance between the cofactors, NAD+ and NADPH, which will play a crucial role in the production of cortisol and corticosterone, may also be affected when Angoras are put under metabolic stress. This will further compound the animal’s inability to sufficiently raise glucocorticoid levels.

Literature Cited Cheng, S. C., and B. W. Harding. 1973. Substrate-induced difference spectral, electron paramagnetic resonance, and enzymatic properties of cholesterol-depleted mitochondrial cytochrome P450 of bovine adrenal cortex. J. Biol. Chem. 248(20):7263−7271. Conley, A. J., and I. M. Bird. 1997. The role of cytochrome P450 17α-hydroxylase and 3β-hydroxysteroid dehydrogenase in the integration of gonadal and adrenal steroidogenesis via the ∆5 and ∆4 pathways of steroidogenesis in mammals. Biol. Reprod. 56:789−799. Fevold, H. R., P. L. Wilson, and S. M. Slanina. 1978. ACTH-stimulated rabbit adrenal 17α-hydroxylase kinetic properties and a comparison with those of 3β-hydroxysteroid dehydrogenase. J. Steroid. Biochem. 9:1033−1041. Ford, H. C., and L. L. Engel. 1974. Purification and properties of the ∆5-3β-hydroxysteroid dehydrogenase-isomerase system of sheep adrenal cortical microsomes. J. Biol. Chem. 249(5):1363−1368. Geller, D. H., R. J. Auchus, B. B. Mendonca, and W. L. Miller. 1997. The genetic and functional basis of isolated 17,20-lyase defficiency. Nat. Genet. 17(2):201−205.

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Engelbrecht and Swart

Hiwatashi, A., and Y. Ichikawa. 1981. Purification and reconstitution of the steroid 21-hydroxylation system (cytochrome P-450linked mixed function oxidase system) of bovine adrenocortical microsomes. Biochim. Biophys. Acta 664:33−48. Kater, C. E., and E. G. Biglieri. 1994. Disorders of steroid 17 alphahydroxylase deficiency. Endocrinol. Metab. Clin. North Am. 23(2):341−357. Kominami, S., H. Ochi, Y. Kobayashi, and S. Takemori. 1980. Studies on the steroid hydroxylation system in adrenal cortex microsomes. J. Biol. Chem. 255:3386−3394. Lambeth, J. D., H. Kamin, and D. W. Seybert. 1980. Phosphatidylcholine vesicle reconstituted cytochrome P-450SCC. Role of the membrane in control of activity and spin state of the cytochrome. J. Biol. Chem. 255:8282−8288. Miller, W. L., R. J. Auchus, and D. H. Geller. 1997. The regulation of 17,20 lyase activity. Steroids 62(1):133−142. Monno, S., Y. Mizushima, N. Toyoda, T. Kashii, and M. Kobayashi. 1997. A new variant of the cytochrome P450c17(CYP17) gene mutation in three patients with 17 alpha-hydroxylase deficiency. Ann. Hum. Genet. 61(Pt3):275−279. Monno, S., H. Ogawa, T. Date, M. Fujioka, W. L. Miller, and M. Kobayashi. 1994. Mutation of histidine 373 to leucine in cytochrome P450c17 causes 17 alpha-hydroxylase deficiency. J. Biol. Chem. 268(34):25811−25817. Omura, R., and T. Sato. 1964. The carbon monoxide-binding pigment of liver microsomes. J. Biol. Chem. 239:2379−2385. Pierce, J., and C. M. Suelter. 1977. An evaluation of the Coomassie Brilliant Blue G-250 dye-binding method for quantitive protein determination. Anal. Biochem. 81:478−480. Sasano, H., J. I. Mason, and N. Sasano. 1989. Immunohistochemical analysis of cytochrome P-450 17α-hydroxylase in pig adrenal cortex, testis and ovary. Mol. Cell. Endocrinol. 62:197−202. Shinzawa, K., S. Kominami, and S. Takemori. 1985. Studies on cytochrome P-450 (P-45017α, lyase) from guinea pig adrenal microsomes. Dual function of a single enzyme and effect of cytochrome b5. Biochim. Biophys. Acta 833:151−160. Smith, P. K., R. D. Krohn, G. T. Hennanson, A. K. Mallia, F. H. Gartner, M. D. Provenzano, B. K. Fujimoto, N. M. Goeke, B. J. Olson, and D. C. Klenk. 1985. Measurement of protein using bicinchonic acid. Anal. Biochem. 150:76−85.

Snyman, M. A., and J. J. Olivier. 1996. Genetic parameters for body weight, fleece weight and fibre diameter in South-African Angora goats. Livest. Prod. Sci. 47(1):1−6. Strott, D. I. 1989. Immunoblotting and dot blotting. J. Immunol. Methods 119:153−187. Suzuki, T., H. Sasano, T. Sawai, J. I. Mason, and H. Nagura. 1992. Immunohistochemistry and in situ hybridization of P-45017α (17α-hydroxylase/17,20-Lyase). J. Histochem. Cytochem. 40(7):903−908. Suzuki, Y., T. Nagashima, Y. Nomura, K. Onigata, K. Nagashima, and A. Morikawa. 1998. A new compound heterozygous mutation (W17X, 436 + 5G -- >T) in the cytochrome P450c17 gene causes 17 alpha-hydroxylase/17,20-lyase deficiency. J. Clin. Endocrinol. Metab. 83(1):199−202. Swart, P., Y. Engelbrecht, D. U. Bellstedt, C. A. de Villiers, and C. Dreesbeimdieke. 1995. The effect of cytochrome b5 on progesterone metabolism in the ovine adrenal. Endocr. Res. 21(12):297−306. Swart, P., Y. Engelbrecht, and T. Herselman. 1996. An investigation of hypo-adrenocorticism in Angora goats. Endocr. Res. 22(4):563−565. Swart, P., P. C. Todres, A. C. Swart, and K. J. van der Merwe. 1988. Micro-assay for sheep 11 beta-hydroxylase activity using highperformance liquid chromatography for steroid analysis. J. Chromatogr. 442:424−430. Takemori, S., and S. Kominami. 1984. The role of cytochromes P-450 in adrenal steroidogenesis. Trends Biochem. Sci. 9:393−396. Tuckey, R. C., and K. J. Cameron. 1993. Side-chain specificities of human and bovine cytochrome P-450SCC. Eur. J. Biochem. 217:209−215. Van Rensburg, S. J. 1973. Reproductive physiology and endocrinology of normal and habitually aborting Angora goats. Onderstepoort J. Vet. Res. 38:1−62. Weber, K., and M. Osborn. 1969. The reliability of molecular weight determinations by dodecyl sulfate polyacrylamide gel electrophoresis. J. Biol. Chem. 244:4406−4412. Wentzel, D. 1987. Effects of nutrition on reproduction in the Angora goat. Proceedings of the IV International Conference on Goats, Brazilia, Brazil. p 571 (Abstr.). Wentzel, D., K. S. Viljoen, and L.J.J. Botha. 1979. Physiological and endocrinological reactions to cold stress in the Angora goat. Agroanimalia 11:19.