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-3ß, 20α-diol (3ß,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,20ßDHP. The concentrations of circulating pregnanes in periparturient mares were (highest to lowest) 3α,20ßDHP 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,20ßDHP were significantly less biopotent. At prepartum concentrations, both 20αDHP and 3α,20ßDHP 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.

Porcine hypothalamic aromatase cytochrome P450: isoform characterization, sex-dependent activity, regional expression, and regulation by enzyme inhibition in neonatal boars.

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.

5α-dihydroprogesterone concentrations and synthesis in non-pregnant mares.

In vivo and in vitro evidence indicates that the bioactive, 5α-reduced progesterone metabolite, 5α-dihydroprogesterone (DHP) is synthesized in the placenta, supporting equine pregnancy, but its appearance in early pregnancy argues for other sites of synthesis also. It remains unknown if DHP circulates at relevant concentrations in cyclic mares and, if so, does synthesis involve the non-pregnant uterus? Jugular blood was drawn daily from cyclic mares (n = 5). Additionally, ovariectomized mares (OVX) and geldings were administered progesterone (300 mg) intramuscularly. Blood was drawn before and after treatment. Incubations of whole equine blood and hepatic microsomes with progesterone were also investigated for evidence of DHP synthesis. Sample analysis for progesterone, DHP and other steroids employed validated liquid chromatography-tandem mass spectrometry methods. Progesterone and DHP appeared a day (d) after ovulation in cyclic mares, was increased significantly by d3, peaking from d5 to 10 and decreased from d13 to 17. DHP was 55.5 ± 3.2% of progesterone concentrations throughout the cycle and was highly correlated with it. DHP was detected immediately after progesterone administration to OVX mares and geldings, maintaining a relatively constant ratio with progesterone (47.2 ± 2.9 and 51.2 ± 2.7%, respectively). DHP was barely detectable in whole blood and hepatic microsome incubations. We conclude that DHP is a physiologically relevant progestogen in cyclic, non-pregnant mares, likely stimulating the uterus, and that it is synthesized peripherally from luteal progesterone but not in the liver or blood. The presence of DHP in pregnant perissodactyla as well as proboscidean species suggests horses may be a valuable model for reproductive endocrinology in other exotic taxa.

Placental expression and molecular characterization of aromatase cytochrome P450 in the spotted hyena (Crocuta crocuta).

At birth, the external genitalia of female spotted hyenas (Crocuta crocuta) are the most masculinized of any known mammal, but are still sexually differentiated. Placental aromatase cytochrome P450 (P450arom) is an important route of androgen metabolism protecting human female fetuses from virilization in utero. Therefore, placental P450arom expression was examined in spotted hyenas to determine levels during genital differentiation, and to compare molecular characteristics between the hyena and human placental enzymes. Hyena placental P450arom activity was determined at gestational days (GD) 31, 35, 45, 65 and 95 (term, 110), and the relative sensitivity of hyena and human placental enzyme to inhibition by the specific inhibitor, Letrozole, was also examined. Expression of hyena P450arom in placenta was localized by immuno-histochemistry, and a full-length cDNA was cloned for phylogenetic analysis. Aromatase activity increased from GD31 to a peak at 45 and 65, apparently decreasing later in gestation. This activity was more sensitive to inhibition by Letrozole than was human placental aromatase activity. Expression of P450arom was localized to syncytiotrophoblast and giant cells of mid-gestation placentas. The coding sequence of hyena P450arom was 94% and 86% identical to the canine and human enzymes respectively, as reflected by phylogenetic analyses. These data demonstrate for the first time that hyena placental aromatase activity is comparable to that of human placentas when genital differentiation is in progress. This suggests that even in female spotted hyenas clitoral differentiation is likely protected from virilization by placental androgen metabolism. Decreased placental aromatase activity in late gestation may be equally important in allowing androgen to program behaviors at birth. Although hyena P450arom is closely related to the canine enzyme, both placental anatomy and P450arom expression differ. Other hyaenids and carnivores must be investigated to determine the morphological and functional ancestral state of their placentas, as it relates to evolutionary relationships among species in this important taxonomic group.

Equine 5α-reductase activity and expression in epididymis.

The 5α-reductase enzymes play an important role during male sexual differentiation, and in pregnant females, especially equine species where maintenance relies on 5α-reduced progesterone, 5α-dihydroprogesterone (DHP). Epididymis expresses 5α-reductases but was not studied elaborately in horses. Epididymis from younger and older postpubertal stallions was divided into caput, corpus and cauda and examined for 5α-reductase activity and expression of type 1 and 2 isoforms by quantitative real-time polymerase chain reaction (qPCR). Metabolism of progesterone and testosterone to DHP and dihydrotestosterone (DHT), respectively, by epididymal microsomal protein was examined by thin-layer chromatography and verified by liquid chromatography tandem mass spectrometry (LC-MS/MS). Relative inhibitory potencies of finasteride and dutasteride toward equine 5α-reductase activity were investigated. Pregnenolone was investigated as an additional potential substrate for 5α-reductase, suggested previously from in vivo studies in mares but never directly examined. No regional gradient of 5α-reductase expression was observed by either enzyme activity or transcript analysis. Results of PCR experiments suggested that type 1 isoform predominates in equine epididymis. Primers for the type 2 isoform were unable to amplify product from any samples examined. Progesterone and testosterone were readily reduced to DHP and DHT, and activity was effectively inhibited by both inhibitors. Using epididymis as an enzyme source, no experimental evidence was obtained supporting the notion that pregnenolone could be directly metabolized by equine 5α-reductases as has been suggested by previous investigators speculating on alternative metabolic pathways leading to DHP synthesis in placenta during equine pregnancies.

Structural analysis of the gene encoding human 3 beta-hydroxysteroid dehydrogenase/delta 5----4-isomerase.

The structural gene encoding human 3 beta-hydroxysteroid dehydrogenase/delta 5----4-isomerase (3 beta HSD) was isolated from a human EMBL3 genomic library. The gene encompasses approximately 8 kilobases of DNA and is comprised of two large introns and three exons encoding amino acid residues 1-48, 49-103, and 104-373, respectively. The exonic sequence is identical to that of the cDNA that we previously isolated and expressed in COS 1 cells. DNA sequence analysis reveals a putative TATA (TATATAA) motif 26 basepairs up-stream of the beginning of exon I, as determined by S1 nuclease protection analysis. However, primer extension analysis using poly(A)+ RNA isolated from both placenta and corpora lutea indicates that the RNA initiates up-stream of the putative TATA motif, and that an additional 53-basepair exon, which is untranslated, is present 5' to the first coding exon. Southern hybridization analysis of genomic DNA using a single exon probe suggests that there may be more than one copy of the gene in the human genome. In addition, we confirm from Southern analysis of genomic DNA isolated from human x hamster somatic cell hybrids that the gene is located on human chromosome 1. These findings will provide a foundation for the characterization of apparent 3 beta HSD clinical deficiencies when these are due to a mutation in the structural gene.

Functional domains of human aromatase cytochrome P450 characterized by linear alignment and site-directed mutagenesis.

The relationship of function to structure of aromatase cytochrome P450 (P450arom; the product of the CYP19 gene) has been examined by means of sequence alignment and site-directed mutagenesis. Comparison has been made between the sequence of P450arom and the two soluble bacterial cytochrome P450 isoforms, whose three-dimensional structure has been determined (P450BM3 and P450cam). From this comparison, it appears that although there is a similarity of overall structure in cytochromes P450, there is enough significant difference in the regions involved in substrate recognition and substrate binding that residues believed to be involved, even in the known structures, must be tested. With this in mind, we have generated a detailed alignment of P450arom, including the definition of putative alpha-helices and beta-sheets based on comparison of the alignments of P450BM3 and P450cam, generated from their three-dimensional structure, and have made mutations in regions we believe to be involved in substrate recognition at the solvent surface and orientation in the heme pocket. We have mutated F116 and F134 to determine if they are present in the heme pocket, and Q225 and L228 to determine if they are a part of the substrate recognition loop. Although F116E is essentially inactive and may be a folding mutant or may inhibit reductase binding, F134E is more active than the wild type and may be located in the heme pocket facilitating the hydrogen abstraction from C2 of androstenedione. Mutations at Q225 and L228 also result in the anticipated changes in the apparent Km and maximum velocity.(ABSTRACT TRUNCATED AT 250 WORDS)

Unique regulation of CYP17 expression in the trophectoderm of the preattachment porcine blastocyst.

Expression of the gene encoding cytochrome P450 17alpha-hydroxylase, CYP17, is necessary for adrenal and gonadal steroidogenesis in most species. However, some animals, such as the pig, express CYP17 in the trophectoderm of the preattachment blastocyst, an event associated with estrogen synthesis and the establishment of pregnancy. How trophoblastic expression of CYP17 is regulated in the porcine blastocyst remains unknown and forms the basis of the following studies. The porcine CYP17 gene, including the complete coding and several kilobases of 5'-flanking regions, was cloned and sequenced. Blastocysts were examined by Northern analysis to verify the level of CYP17 transcript, and tissue-specific expression in the trophectoderm was confirmed by in situ hybridization. Primer extension, S1 nuclease protection, and 5'-rapid amplification of cDNA ends confirmed a common proximal transcription start site in adrenals and gonads (-48 bp) but identified a unique distal start site used in porcine trophectoderm (-182 bp). Additionally, reporter analysis of the CYP17 regulatory region demonstrated that constructs (-27 to -718 bp) were unresponsive to forskolin when expressed in porcine trophoblast cells, suggesting that trophoblast may not be able to respond to cAMP induction of this gene. The identification of this distal, previously undescribed, transcriptional start site suggests that unique mechanisms control the expression of CYP17 in porcine trophectoderm and possibly other genes important in implantation and early placental development.

Growth factors suppress and phorbol esters potentiate the action of dibutyryl adenosine 3',5'-monophosphate to stimulate aromatase activity of human adipose stromal cells.

In previous studies, we observed that the stimulatory effect of (Bu)2cAMP on aromatase activity of human adipose stromal cells was markedly attenuated when fetal calf serum was present in the culture medium. To determine whether growth factors may be the inhibitors of (Bu)2cAMP-stimulated aromatase activity in serum, the effects of growth factors and phorbol esters on aromatase activity of human adipose stromal cells in monolayer culture were investigated. Epidermal growth factor (EGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF) were all without effect on aromatase activity when added by themselves, but markedly inhibited aromatase activity stimulated by (Bu)2cAMP. On the other hand, nerve growth factor, multiplication-stimulating activity, relaxin, and insulin had no effect on aromatase activity, either by themselves or in the presence of (Bu)2cAMP. Thus, EGF, PDGF, and FGF can mimic the inhibitory action of fetal calf serum on (Bu)2cAMP-stimulated aromatase activity of these cells. By contrast, none of these substances was capable of mimicking the effect of serum to facilitate the stimulatory action of dexamethasone on aromatase activity of these cells. The phorbol esters phorbol-12-myristate-13-acetate, phorbol-12,13-didecanoate, and phorbol-12,13-diacetate were also capable of facilitating the action of (Bu)2cAMP to stimulate aromatase activity, but had little or no action on dexamethasone-stimulated aromatase activity or when added by themselves. It is concluded that aromatase is under multifactorial regulation in human adipose stromal cells. The activity is induced by glucocorticoids and by agents that stimulate cAMP-dependent protein kinase; the latter effect is potentiated by factors that stimulate protein kinase C, but is suppressed by growth factors such as EGF, FGF, and PDGF, whose actions are believed to be mediated by receptor-linked tyrosine kinase activity.

Isolation and characterization of a complementary deoxyribonucleic acid insert encoding bovine aromatase cytochrome P450.

Aromatase, an enzyme complex comprised of aromatase cytochrome P450 (P450arom; the product of the CYP19 gene) and the flavoprotein NADPH-cytochrome P450 reductase, catalyzes the conversion of androgens to estrogens. Three cDNA inserts encoding P450arom were isolated from a bovine placental cDNA library. These inserts were sequenced and found to correspond closely to human P450arom sequence from the internal EcoRI restriction site (exon III) through the termination codon (exon X) into the 3'-untranslated region. The rapid amplification of cDNA ends technique was used to generate the rest of the cDNA 5' of the internal EcoRI site, using mRNA obtained from bovine granulosa cells as a template. This insert was sequenced, and when aligned with the other inserts, an open reading frame was found which was predicted to encode a protein of 503 amino acid residues. The deduced polypeptide shares 84% identity with human P450arom and 79%, 76%, 71%, and 57% identity with mouse, rat, chicken, and trout P450arom, respectively. A full-length open reading frame was generated using the polymerase chain reaction and mRNA obtained from bovine granulosa cells as template. After this insert was ligated into the pCMV5 expression vector, it was transfected into COS-1 monkey kidney tumor cells. We were able to demonstrate aromatase activity by assaying the incorporation of tritium into [3H] water from [1 beta-3H]androstenedione. Northern analysis revealed a single transcript of approximately 6 kilobases in poly(A)+ RNA obtained from bovine placental tissue and granulosa cells. This indicated for the first time a correspondence between the pattern of estrogen biosynthesis throughout the bovine ovarian cycle and the levels of transcripts encoding P450arom. In addition, weak hybridization was noted to transcripts of the expected size, namely 3.4 and 2.9 kilobases, in poly(A)+ RNA obtained from human placental tissue. The large size of the bovine transcript is due to a long 3'-untranslated region, because, based on the rapid amplification of cDNA ends technique, there appeared to be approximately 150 basepairs 5' of the start site of translation, and we were never able to find a polyadenylation site, even in one clone that went well past the corresponding polyadenylation site in human P450arom.

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 microM) inhibited more than 97% of this activity, whereas another imidazole, etomidate (1 microM), 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 of other steroid metabolites accumulating in the media of cell lines expressing either isozyme. Tritiated water formed during aromatization of substrates 3H labeled at the C1 and C2 positions was stereo-selective for the beta orientation, but less so for testosterone than androstenedione during metabolism by the porcine placental (and human) isozyme than the gonadal isozyme. Testosterone showed a higher affinity for the porcine placental P450arom than the gonadal P450arom, but both isozymes had similar affinities for androstenedione. Testosterone was also aromatized more slowly than androstenedione by the porcine gonadal P450arom. These data suggest that catalytic differences have arisen in the substrate binding pocket during the evolution of isozymes of porcine P450arom that affect androgen metabolism, particularly the aromatization of testosterone. The physiological significance of these differences to the reproductive biology of the pig remains to be determined.

Regulation of microsomal P450, redox partner proteins, and steroidogenesis in the developing testes of the neonatal pig.

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), 17alpha-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 b(5) 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 b(5) 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 b(5) 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 b(5) expression appears to be constitutive, but reductase levels saturate both P450c17 and P450arom.

Cytochrome p450-dependent lipid metabolism in preovulatory follicles.

Estrogen biosynthesis and proteolysis are both important processes involved in ovarian follicular development, which may be influenced by cytochrome P450 (CYP)-dependent fatty acid metabolites. However, CYP-dependent lipid metabolism has not been characterized with respect to follicular maturation in vivo. Therefore, follicular fluid was collected in the hours before and after the LH surge in pigs, and concentrations of epoxy, hydroxy, and dihydroxy lipids were measured by liquid chromatography tandem mass spectrometry. Arachidonate oxidation and epoxyeicosatrienoic acid hydrolysis to dihydroxyeicosatrienoic acids (DHETs) were also assessed in thecal and granulosa tissue fractions, and the expression of CYP epoxygenases was evaluated by immunoblots using available antisera. To evaluate soluble epoxide hydrolase (sEH) expression, the porcine sEH was cloned from ovarian tissue, expressed and purified for antibody generation. The follicular fluid oxylipin concentrations ranged from 1-150 nm depending on the compound and estrous stage. The follicular fluid concentrations of CYP-dependent oxylipins increased at estrus, as did sEH expression; however, significant changes in epoxides were not observed, and the 11,12-DHET peak was delayed. The ratio of 14,15-DHET:11,12-DHET across all samples correlated with the log of follicular fluid estradiol concentrations (P < 0.01). Epoxygenase activities were similar in theca and granulosa, varying little with follicular development, whereas the decline of a single CYP2J isoform at ovulation was observed by immunoblots. The sEH activity was higher in granulosa than in theca. Finally, the dynamic changes in follicular CYP-dependent arachidonic acid metabolites and their modulatory function in vascular models suggest roles for these metabolites in follicular maturation, which may include regulation of estradiol biosynthesis and preovulatory remodeling of the follicular wall that should be fully explored in future studies.

The primate adrenal zona reticularis is defined by expression of cytochrome b5, 17alpha-hydroxylase/17,20-lyase cytochrome P450 (P450c17) and NADPH-cytochrome P450 reductase (reductase) but not 3beta-hydroxysteroid dehydrogenase/delta5-4 isomerase (3beta-HSD).

Biochemical studies suggest that 17,20-lyase activity, and thus efficient synthesis of androgens by human P450c17, requires both reductase and the accessory protein cytochrome b5. Since the human and primate zona reticularis (ZR) secrete androgens, the expression of these proteins, and of 3beta-HSD, was investigated by immunocytochemistry in the adrenal cortex of the mature rhesus macaque. Cytochrome b5 expression was restricted to the cells of the ZR which appeared deficient in 3beta-HSD. However, both P450c17 and reductase were evident throughout the zona fasciculata. These data provide essential evidence in support of a functional role for cytochrome b5 in the regional control of 17alpha-hydroxylase and 17,20-lyase activities of P450c17 and thereby adrenal C19 steroid secretion by the primate adrenal gland.

Functional ovarian and placental isoforms of porcine aromatase.

Functional isoforms of porcine aromatase cytochrome P-450 were cloned from placenta, and ovarian theca interna and granulosa tissues, and full length cDNAs were expressed in vitro. Porcine theca and granulosa expressed an identical form of P-450arom. This ovarian cDNA encoded for a protein of 501 amino acids, two amino acids shorter at the N-terminal end than placental P-450arom isoform (503 residues). Overall, the two isoforms exhibited 93% nucleotide and 87% amino acid identity with each other, and both were highly homologous, at the nucleotide and amino acid levels, to human and bovine P-450arom, also 503 amino acid proteins. Analysis of the predicted amino acid sequence further suggested that the regions of the cDNAs, corresponding to presumed exons III, V and IX, assuming conservation of intron-exon boundaries with the human P-450arom gene, were conserved in the porcine placental and ovarian enzymes, while sequence variance occurred in all other putative exons. In vitro expression indicated that the cDNA encoding porcine placental P-450arom was almost 10-fold more active in the synthesis of estrone from androstenedione than was the ovarian isoform which synthesized more 19OH-androstenedione than estrone. Western analysis of transfected Cos1 cells suggested that the differences in activity were not due to levels of expression of the cDNAs since similar levels of immunodetectable protein were observed in cells transfected with each construct. Both isoforms were sensitive to inhibition of activity by the specific aromatase inhibitors, 4OH-androstenedione and CGS16949A. In addition, activity of the enzyme encoded by the ovarian P-450arom cDNA was suppressed by etomidate, an inhibitor of cytochrome P-450 11beta-hydroxylase, but the placental P-450arom isoform was not. These functional differences were consistent with observations made in similar experiments involving P-450arom activity in freshly homogenized tissues. These data provide evidence of the existence of distinct, intraspecies isoforms of P-450arom, the first described in any species and suggest that pigs possess a unique mechanism for regulating androgen metabolism.

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.

Sequencing of cDNA inserts encoding aromatase cytochrome P-450 (P-450AROM).

Two cDNA inserts complementary to mRNA encoding aromatase cytochrome P-450 (P-450AROM) have been isolated and characterized by restriction mapping and sequencing. The overlapping sequence encoded by these inserts is identical, and a putative heme-binding region has been identified. In addition, the open reading frame contains the sequences of all four cysteine-containing tryptic peptides isolated by Chen et al. (1986) from purified cytochrome P-450AROM. The inserts differ in the use of alternative poly A-addition signals, which is consistent with the presence of two major species of mRNA in human placenta, of 3.0 and 2.4 kb, which hybridize to these inserts. The identity of sequence between the two inserts and the likely presence of alternative poly A-addition signals, is suggestive that only one form of cytochrome P-450AROM is encoded by these mRNA species.

A comparative approach to structure-function studies of mammalian aromatases.

To date, structure--function studies of aromatase cytochrome P450 (P450arom) have been advanced by point mutation analyses utilizing almost exclusively the human enzyme, in conjunction with computer-generated models of the three-dimensional form of the enzyme based on prokaryotic cytochromes P450. Recent studies have identified duplicated isozymes of porcine P450arom, the gonadal and placental forms of which appear to differ substantially in substrate utilization and inhibitor sensitivity. We present a comparative approach to define regions of P450arom responsible for specific functional characteristics using complimentary DNAs encoding the porcine isozymes. Constructs encoding the native and chimeric porcine and human P450arom enzymes were transiently expressed and activity was assessed using the tritiated water assay. Sensitivity to inhibition by the imidazole etomidate was investigated, and P450arom expression was assessed by immunoblot analysis. All constructs yielded active P450arom, suggesting that exchanging entire structural elements does not preclude catalytic function. The activity of the gonadal isozyme was shown to be inhibited by etomidate at concentrations 185 and 300-fold lower than those required to induce a similar inhibition of the placental and human enzymes, respectively. In contrast, there was only a two-fold difference in the sensitivity of the gonadal and placental isozymes to inhibition by CGS16949A. Analysis of chimeric constructs indicated that the sensitivity to etomidate was associated with residues in the B, B' and C helices of the gonadal P450arom encompassing only one of six putative substrate recognition sites. Additionally, sensitivity to etomidate was not correlated with enzyme activity among the chimeric enzymes. Therefore, it appears that residues of the porcine gonadal P450arom that are responsible for etomidate binding may be distinct from those involved in substrate recognition and metabolism. These data support the notion that a comparative approach employing the use of chimeric enzymes provides a useful tool in directing point mutational analysis to determine residues important in inhibitor and perhaps substrate recognition of P450 enzymes such as P450arom. These studies are currently in progress.

Tissue-specific promoters regulate aromatase cytochrome P450 expression.

In the human, estrogen biosynthesis occurs in several tissue sites, including ovary, placenta, adipose, and brain. Recent work from our laboratory has indicated that tissue-specific expression of aromatase cytochrome P450 (P450arom), the enzyme responsible for estrogen biosynthesis, is determined, in part, by the use of tissue-specific promoters. Thus the expression of P450arom in human ovary appears to utilize a promoter proximal to the translation start-site. This promoter is not utilized in placenta but instead, the promoter used to drive aromatase expression in placenta is at least 40 kb upstream from the translational start-site. In addition, there is a minor promoter used in the expression of a small proportion of placental transcripts which is 9 kb upstream from the start of translation. Transcripts from these promoters are also expressed in other fetal tissues including placenta-related cells such as JEG-3 choriocarcinoma cells, hydatidiform moles, and other fetal tissues such as fetal liver. On the other hand, in adipose tissue expression of P450arom may be achieved by yet another, adipose-specific promoter. The various 5'-untranslated exons unique for expression driven by each of these promoters are spliced into a common intron/exon boundary upstream from the translational start-site. This means that the protein expressed in each of the various tissue-specific sites of estrogen biosynthesis is identical.

Sex identification in mammals with polymerase chain reaction and its use to examine sex effects on diameter of day-10 or -11 pig embryos.

The objectives of this study were to develop a rapid method for sex determination for several mammalian species using polymerase chain reaction (PCR) and to use this method to determine whether there is a significant developmental difference in spherical diameter between male and female d-10 or -11 porcine embryos. The PCR system was developed and verified using genomic DNA from pigs of known sex, then it was tested with genomic DNA from several other mammalian species. Sex is determined by amplification of two genes in a single reaction. The presence or absence of a region of the Sry (sex-determining region Y) gene determines sex, and amplification of the Zfy (male) or Zfx (female) genes acts as a positive control for PCR. Sex determination was successful for all animals tested, including pigs, cattle, sheep, goats, llamas, horses, humans, baboons, dogs, cats, rats, and mice. A total of 209 embryos were collected from 21 crossbred gilts on d 10 or 11 of gestation, and their diameters were measured. No significant difference in embryo diameter was detected between male and female embryos, indicating that sexual dimorphism in embryonic growth in pigs does not occur before the period of rapid embryo elongation. The present sexing technique using PCR is rapid (approximately 6 h from receipt of embryos to results), and it may be useful for examining the effects of sex on any trait of interest in early porcine embryos and embryos from several other mammals.