The isolation and cloning of a full-length cDNA insert complementary to mRNA encoding human aromatase system cytochrome P-450 is reported. The insert contains an open reading frame encoding a protein of 503 amino acids. This gene is clearly a member of the cytochrome P450 gene superfamily, because the sequence contains regions of marked homology to those of other members, notably a putative membrane-spanning region, I helix, Ozols, and hemebinding regions. The cDNA was inserted into a modified pCMV vector and expressed in COS-1 monkey kidney tumor cells. The expressed protein was similar in size to human placental aromatase system cytochrome P-450, as detected by immunoblot analysis, and catalyzed the aromatization of androstenedione, testosterone, and 16a-hydroxyandrostenedione. This activity was inhibited by the known aromatase inhibitors, 4-hydroxyandrostenedione and econazole. Thus the several steps involved in the aromatization reaction appear to be catalyzed by a single polypeptide chain, which can metabolize the three major physiological substrates.

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.

One of the most widely accepted axioms of mammalian reproductive biology is that pregnancy requires the (sole) support of progesterone, acting in large measure through nuclear progesterone receptors (PRs) in uterine and cervical tissues, without which pregnancy cannot be established or maintained. However, mares lack detectable progesterone in the latter half of pregnancy. Instead of progesterone, several (mainly 5α-reduced) pregnanes are elevated and have long been speculated to provide progestational support in lieu of progesterone itself. To the authors' knowledge, evidence for the bioactivity of a second potent endogenously synthesized pregnane able to support pregnancy in the absence of progesterone has never before been reported. The 5α-reduced progesterone metabolite dihydroprogesterone (DHP) was shown in vivo to stimulate endometrial growth and progesterone-dependent gene expression in the horse at subphysiological concentrations and to maintain equine pregnancy in the absence of luteal progesterone in the third and fourth weeks postbreeding. Results of in vitro studies indicate that DHP is an equally potent and efficacious endogenous progestin in the horse but that the PR evolved with increased agonistic potency for DHP at the expense of potency toward progesterone based on comparisons with human PR responses. Sequence analysis and available literature indicate that the enzyme responsible for DHP synthesis, 5α-reductase type 1, also adapted primarily to metabolize progesterone and thereby to serve diverse roles in the physiology of pregnancy in mammals. Our confirmation that endogenously synthesized DHP is a biopotent progestin in the horse ends decades of speculation, explaining how equine pregnancies survive without measurable circulating progesterone in the last 4 to 5 mo of gestation.ince first crystallized almost eight decades ago, progesterone has remained the only endogenous member of the progestin class of steroids defined by its singular ability to maintain pregnancy (1), acting, in large measure, through nuclear progesterone receptors (PRs) in uterine and cervical tissues, without which pregnancy cannot be established or maintained (2). Birth is thought to be triggered by a decrease in systemic progesterone concentrations (withdrawal) (3), even though this is not evident in mares (4), women, or guinea pigs (5), a disparity that limits the utility of other animal models for preterm labor (6). The vast majority of studies have focused on measuring progesterone, with most using immunoassays that necessarily cross-react with multiple pregnanes (7), the bioactivity of which remain uncharacterized. This is reasonable because, in contrast to androgens, estrogens, and corticoids, for which multiple natural biopotent analogs are known, no other endogenous pregnane has ever been shown to substitute for progesterone in pregnancy in any mammal.However, over five decades ago, Short (8) reported that circulating progesterone concentrations in pregnant mares were surprisingly low at <4 ng/mL, as did Holtan e...

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The expression of aromatase cytochrome P450 (P450arom) in the adrenal glands, testes, and placentas of fetal and newborn pigs was investigated. Western immunoblot analysis detected a single 48-50-kDa protein band in these tissues as well as in other porcine tissues known to express P450arom including Day 12 tubular conceptuses, theca interna, and granulosa. Slight differences in migration suggested that the P450arom protein expressed in the testis was larger than that in the adrenal gland, which was, in turn, larger than that in placenta, theca, and granulosa. Consistent with P450arom expression in these tissues, a cDNA encoding porcine P450arom hybridized to a 2.3-kb transcripts in Northern analyses of porcine blastocysts, placentas, and fetal and newborn adrenal glands and testes, as well as in theca and granulosa tissues from preovulatory follicles. No differences in transcript size were detectable among tissues. The identity of P450arom transcripts was confirmed by sequence analysis of partial cDNA clones amplified from porcine fetal adrenal glands, testes, and placentas according to the RACE procedure. The sequences of the adrenal and testis clones were identical but differed from the placental sequence, which represented the first 85 amino acids of porcine P450arom. Specifically, the adrenal and testis clones expressed transcripts that resembled the ovarian isoform of porcine P450arom, rather than the porcine placental isoform, predicting a two-amino acid deletion and 12 predicted amino acid substitutions. P450arom activity was examined to further define expression in these tissues. Activity in adrenal, testis, and placental homogenates was inhibited by 4-hydroxyandrostenedione (4OH-A4) whereas inhibition by etomidate was demonstrated in the adrenal and testis homogenates but not in the placental homogenates. The sensitivity of activity in the newborn porcine adrenal glands and testes to inhibition by etomidate was similar to that of ovarian P450arom activity. The level of P450arom activity was highest in the placenta and lowest in the adrenal gland, and no effect of fetal sex was noted in either tissue. Immunocytochemical studies localized the expression of P450arom in the adrenal gland of newborns to cells at the corticomedullary junction and, with greater intensity, to cells around the developing medullary lobules. The same cells expressed cytochrome P450 17 alpha-hydroxylase, the expression of which also extended throughout the zona fasciculata. The interstitial cells were the site of P450arom expression in the testis, but no expression could be detected in any cells with the spermatic tubules. These data demonstrate that fetal and newborn porcine adrenal glands and testes express an active P450arom that resembles the isoform expressed in the ovary. The localization of adrenal expression suggests a possible role in medullary maturation and function in the fetal and newborn pig.

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Mounting evidence underscores the importance of proteinprotein interactions in the functional regulation of drug-metabolizing P450s, but few studies have been conducted in membrane environments, and none have examined P450s catalyzing sex steroid synthesis. Here we report specific protein-protein interactions for full-length, human, wild type steroidogenic cytochrome P450 (P450, CYP) enzymes: 17␣-hydroxylase/ 17,20-lyase (P450c17, CYP17) and aromatase (P450arom, CYP19), as well as their electron donor NADPH-cytochrome P450 oxidoreductase (CPR). Fluorescence resonance energy transfer (FRET) 3 in live cells, coupled with quartz crystal microbalance (QCM), and atomic force microscopy (AFM) studies on phosphatidyl choline ؎ cholesterol (mammalian) biomimetic membranes were used to investigate steroidogenic P450 interactions. The FRET results in living cells demonstrated that both P450c17 and P450arom homodimerize but do not heterodimerize, although they each heterodimerize with CPR. The lack of heteroassociation between P450c17 and P450arom was confirmed by QCM, wherein neither enzyme bound a membrane saturated with the other. In contrast, the CPR bound readily to either P450c17-or P450arom-saturated surfaces. Interestingly, N-terminally modified P450arom was stably incorporated and gave similar results to the wild type, although saturation was achieved with much less protein, suggesting that the putative transmembrane domain is not required for membrane association but for orientation. In fact, all of the proteins were remarkably stable in the membrane, such that high resolution AFM images were obtained, further supporting the formation of P450c17, P450arom, and CPR homodimers and oligomers in lipid bilayers. This unique combination of in vivo and in vitro studies has provided strong evidence for homodimerization and perhaps some higher order interactions for both P450c17 and P450arom.