C. J. Corbin

Changes in Testosterone Metabolism Associated with the Evolution of Placental and Gonadal Isozymes of Porcine Aromatase Cytochrome P450

Differences in the catalytic activity of the placental and gonadal isozymes of porcine aromatase cytochrome P450 (P450arom) were examined in cell lines exhibiting stable expression of recombinant enzyme. Cell lines were selected that expressed high, but similar, immunodetectable levels of each isozyme based on Western analysis. Aromatase activity varied with growth in culture, decreasing at confluence from a peak reached between 50–80% cell density. Cells expressing the placental isozyme had 3–5 times higher catalytic activity (per mg protein) than those expressing the gonadal isozyme. The P450arom inhibitor fadrazole (1 μm) inhibited more than 97% of this activity, whereas another imidazole, etomidate (1 μm), selectively inhibited gonadal P450arom activity by 92%. Estrogen synthesis from androstenedione and testosterone was determined by RIA and confirmed by HPLC analysis, which also identified the accumulation of the 19-hydroxy and 19-oxo intermediates of the respective substrates. There was no evidence...

2,3,7,8-Tetrachlorodibenzo-p-dioxin decreases estradiol production without altering the enzyme activity of cytochrome P450 aromatase of human luteinized granulosa cells in vitro

Abstract This study was designed to examine the in vitro effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on steroid production in human luteinizing granulosa cells (hLGC). TCDD (10 nM) or its solvent was added at the time of changing medium, directly to the cells, every 48 h for 8 days. To test the hypothesis that TCDD reduces estradiol (E2) synthesis by an effect on cytochrome P450 aromatase, aromatase protein and aromatase activity were evaluated. E2 decreased without changing either aromatase protein or its enzyme activity, suggesting that the target of toxicity of TCDD is upstream of aromatase in the steroidogenic pathway. When hLGC were incubated in the presence of labeled E2, no changes in the metabolism of E2 by treatment were observed. Since TCDD did not change progesterone or 17α-hydroxyprogesterone, the inhibition of E2 synthesis by TCDD would seem not to involve steps such as cholesterol transport. Furthermore, the TCDD effect on E2 concentration in these cells disappeared in the presence of excess androgens. We conclude that the inhibition of E2 secretion by TCDD involves intermediate steps, specifically, the provision of androgens for aromatization.

Costs and Consequences of Cellular Compartmentalization and Substrate Competition among Human Enzymes Involved in Androgen and Estrogen Synthesis

ABSTRACT The impact of compartmental expression of steroidogenic enzymes and of changes in flux through delta5 and delta4 metabolism on sex steroid synthesis was investigated by rebuilding pathways using recombinant enzyme expression by infection of insect cells with recombinant baculovirus constructs. Human cytochromes 17alpha-hydroxylase/17,20-lyase (P450c17) and aromatase (P450arom), always coexpressed with their redox partner NADPH-P450 oxidoreductase (CPR) and 3beta-hydroxysteroid dehydrogenase/delta5-4 isomerase (3betaHSD; types 1 or 2), were compartmentally expressed in different cell populations or coexpressed together with pregnenolone (100 nM) as substrate. Estrone was compared among cell compartments expressing different enzyme combinations or in cells coexpressing all enzymes (experiment 1). Additionally, P450c17, 3betaHSD, and CPR were all coexpressed, and androstenedione was measured in cells with different 3betaHSD expression levels or activity using an inhibitor, trilostane (experiment 2). Steroids were measured by immunoassay and mass spectrometry. In experiment 1, partitioning of P450c17, P450arom, and 3betaHSD markedly decreased estrone synthesis in comparison to cells coexpressing enzymes in different combinations. However, partitioning P450arom with 3betaHSD from P450c17 in different cell populations resulted in more estrone than either of the other two-cell compartment models. In experiment 2 (cells coexpressing P450c17, 3betaHSD, and CPR), androstenedione secretion was (paradoxically) higher at lower levels of 3betaHSD, and partial inhibition of 3betaHSD by trilostane also increased androstenedione when 3betaHSD expression was high. We conclude 1) that tissue or cell-specific, partitioned expression of sex steroid synthesizing enzymes limits rather than maximizes estrogen synthesis and 2) that limiting metabolism by 3betaHSD can paradoxically promote androgen synthesis when 3betaHSD expression is high by promoting delta5-steroid flux.

Biochemical Assessment of Limits to Estrogen Synthesis in Porcine Follicles

Abstract 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 17α-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.

Porcine gonadal and placental isozymes of aromatase cytochrome P450: sub-cellular distribution and support by NADPH-cytochrome P450 reductase.

Functional differences exist between the porcine gonadal and placental aromatase cytochrome P450 (P450arom) isozymes that are encoded by separate genes. The present experiments investigated the sub-cellular location of these isozymes, their dependence on the redox partner protein NADPH-cytochrome P450 reductase (P450 reductase) for catalytic activity and the release of steroid intermediates during the aromatization of androgens to estrogen. After differential centrifugation, similar levels of gonadal and placental porcine P450arom were found along with P450 reductase in the microsomal compartment using activity and immunoblot analyses. Activity was stimulated much more by recombinant P450 reductase addition, and higher levels of 19-hydroxy and 19-oxo intermediates accumulated during androstenedione and testosterone metabolism, in cells expressing the gonadal compared to the placental isozyme. No other steroid products were identified by HPLC. Thus, the porcine gonadal P450arom is more sensitive to P450 reductase deprivation than is the placental P450arom, accounting in part for catalytic differences between these isozymes.

Serum anti-Müllerian hormone concentrations in stallions: Developmental changes, seasonal variation, and differences between intact stallions, cryptorchid stallions, and geldings

Anti-Müllerian hormone (AMH), a homodimeric glycoprotein, is secreted early in fetal life when it exerts a crucial function in sexual differentiation. The secretion of AMH in male humans persists after birth and is characterized by high prepubertal concentrations followed by a significant decrease at the onset of puberty. The expression of AMH in the normal and cryptorchid equine testis is well characterized but data regarding circulating AMH concentrations are lacking. The objectives of this study were to determine serum AMH concentrations in neonatal colts and fillies, prepubertal colts, and postpubertal stallions, and to evaluate variations in serum AMH related to season and gonadal status of stallions. In addition, we examined the presence and determined concentrations of AMH in seminal plasma of mature stallions. Serum AMH concentrations were significantly higher in neonatal colts than in neonatal fillies. Moreover, concentrations of AMH are high in prepubertal colts whereas significantly lower concentrations were detected after puberty. In intact mature stallions, season influenced AMH concentrations with significantly higher concentrations during spring and summer. Serum AMH concentrations were significantly higher in cryptorchid stallions compared with intact stallions or geldings. Finally, AMH was identified in seminal plasma of intact mature stallions, but there was no significant correlation between serum and seminal plasma AMH concentrations. In conclusion, serum AMH concentration varies with sex in the neonatal period, postnatal sexual development and season, and serum AMH concentration can be used as a biomarker for the presence of testicular tissue.

Porcine Hypothalamic Aromatase Cytochrome P450: Isoform Characterization, Sex-Dependent Activity, Regional Expression, and Regulation by Enzyme Inhibition in Neonatal Boars

Abstract Domestic pigs have three CYP19 genes encoding functional paralogues of the enzyme aromatase cytochrome P450 (P450arom) that are expressed in the gonads, placenta, and preimplantation blastocyst. All catalyze estrogen synthesis, but the gonadal-type enzyme is unique in also synthesizing a nonaromatizable biopotent testosterone metabolite, 1OH-testosterone (1OH-T). P450arom is expressed in the vertebrate brain, is higher in males than females, but has not been investigated in pigs, to our knowledge. Therefore, these studies defined which of the porcine CYP19 genes was expressed, and at what level, in adult male and female hypothalamus. Regional expression was examined in mature boars, and regulation of P450arom expression in neonatal boars was investigated by inhibition of P450arom with letrozole, which is known to reprogram testicular expression. Pig hypothalami expressed the gonadal form of P450arom (redesignated the “gonadal/hypothalamic” porcine CYP19 gene and paralogue) based on functional analysis confirmed by cloning and sequencing transcripts. Hypothalamic tissue synthesized 1OH-T and was sensitive to the selective P450arom inhibitor etomidate. Levels were 4-fold higher in male than female hypothalami, with expression in the medial preoptic area and lateral borders of the ventromedial hypothalamus of boars. In vivo, letrozole-treated neonates had increased aromatase activity in hypothalami but decreased activity in testes. Therefore, although the same CYP19 gene is expressed in both tissues, expression is regulated differently in the hypothalamus than testis. These investigations, the first such studies in pig brain to our knowledge, demonstrate unusual aspects of P450arom expression and regulation in the hypothalamus, offering promise of gaining better insight into roles of P450arom in reproductive function.

Adaptive Evolution of Mammalian Aromatases: Lessons From Suiformes

Estrogen synthesis evolved in chordates to control reproduction. The terminal enzyme in the cascade directly responsible for estrogen synthesis is aromatase cytochrome P450 (P450arom) encoded by the CYP19 gene. Mammals typically have a single CYP19 gene but pigs, peccaries and other Suiformes have two or more resulting from duplication in a common ancestor. Duplication of CYP genes in the steroid synthetic cascade has occurred for only one other enzyme, also terminal, 11beta-hydroxylase P450 (P450c11). P450arom and P450c11 share common substrates and even physiological functions as possible remnants from a common P450 progenitor, perhaps an ancestral P450arom, which is supported by phylogenetic analysis. Conserved tissue-specific expression patterns of P450arom paralogs in placenta and gonads of pigs and peccaries suggest how functional adaptation may have proceeded divergently and influenced adopted reproductive strategies including ovulation rate and litter size. Data suggest that the porcine placental paralog evolved catalytically to protect female conceptuses from testosterone produced by male siblings; the gonadal paralog to synthesize a novel, nonaromatizable testosterone metabolite (1OH-testosterone) that may increase ovulation rate. This would represent a coevolution facilitating litter bearing as pigs diverged from peccaries. Evidence of convergence between the peccary CYP19 genes and lower tissue expression may therefore represent initiation of loss of the functional paralogs. Studies on the Suiforme aromatases provide insights into the evolution of the steroidogenic cascade and metabolic pathways in general, how it translates into physiological adaptations (altered reproductive strategies for instance), and how duplicated genes become stabilized or disappear from genomes.

Progestin withdrawal at parturition in the mare

Mammalian pregnancies need progestogenic support and birth requires progestin withdrawal. The absence of progesterone in pregnant mares, and the progestogenic bioactivity of 5α-dihydroprogesterone (DHP), led us to reexamine progestin withdrawal at foaling. Systemic pregnane concentrations (DHP, allopregnanolone, pregnenolone, 5α-pregnane-3β, 20α-diol (3β,20αDHP), 20α-hydroxy-5α-dihydroprogesterone (20αDHP)) and progesterone) were monitored in mares for 10days before foaling (n=7) by liquid chromatography-mass spectrometry. The biopotency of dominant metabolites was assessed using luciferase reporter assays. Stable transfected Chinese hamster ovarian cells expressing the equine progesterone receptor (ePGR) were transfected with an MMTV-luciferase expression plasmid responsive to steroid agonists. Cells were incubated with increasing concentrations (0-100nM) of progesterone, 20αDHP and 3α,20βDHP. The concentrations of circulating pregnanes in periparturient mares were (highest to lowest) 3α,20βDHP and 20αDHP (800-400ng/mL respectively), DHP and allopregnanolone (90 and 30ng/mL respectively), and pregnenolone and progesterone (4-2ng/mL). Concentrations of all measured pregnanes declined on average by 50% from prepartum peaks to the day before foaling. Maximum activation of the ePGR by progesterone occurred at 30nM; 20αDHP and 3α,20βDHP were significantly less biopotent. At prepartum concentrations, both 20αDHP and 3α,20βDHP exhibited significant ePGR activation. Progestogenic support of pregnancy declines from 3 to 5days before foaling. Prepartum peak concentrations indicate that DHP is the major progestin, but other pregnanes like 20αDHP are present in sufficient concentrations to play a physiological role in the absence of DHP. The authors conclude that progestin withdrawal associated with parturition in mares involves cessation of pregnane synthesis by the placenta.

Age and season affect serum testosterone concentrations in cryptorchid stallions

Cryptorchidism, characterised by inguinal and/or abdominal retention of one or both testes, is the most common developmental defect encountered in equine veterinary practice. Most cases present with stallion-like behaviour in males without palpable testes and questionable castration history. Evaluation of serum testosterone concentrations is commonly used to distinguish geldings from cryptorchid stallions without a scrotal testis, but results can be variable and difficult to interpret (Lu 2005, Raś and others 2010). Identifying factors that influence serum testosterone concentrations in cryptorchid stallions can be valuable in the interpretation of assay results. Both season and age are known to have a major effect on testosterone concentrations in intact stallions (Berndtson and others 1974, Raeside 1979, Stewart and Roser 1998), but little is known in cryptorchid stallions, with only one study reporting seasonal variation in serum testosterone concentrations (Cox and others 1988). Additionally, it is possible that serum testosterone concentrations in cryptorchid stallions are influenced by age as the cryptorchid testis is subjected to chronic elevated temperatures which could potentially adversely affect Leydig cell function. However, age-related changes in serum testosterone concentrations in cryptorchid stallions have not, to our knowledge, been described. Therefore, the objective of this study was to evaluate the effect of season and age on serum …