Biomedical subjects

E R Simpson

Publications and source records attributed to E R Simpson.

At least 199 records · Page 11Linked to original sources

PubMed1987

Immunohistochemical localization of aromatase cytochrome P-450 and estradiol dehydrogenase in the syncytiotrophoblast of the human placenta.

Immunohistochemistry employing immunoglobulin G fractions raised against aromatase cytochrome P-450 and antiserum against 17 beta-estradiol dehydrogenase was used to localize these two steroid-converting enzymes in the human placenta. Immunostaining for both enzymes was found exclusively in the syncytiotrophoblast, while the underlying cytotrophoblast and the villus core did not stain. Ultrastructural examination of aromatase cytochrome P-450- and 17 beta-estradiol dehydrogenase-labeled sections disclosed immunoreactive product in the membranes of the endoplasmic reticulum; the nucleus, mitochondria, Golgi apparatus, and secretory granules were free of staining. These findings suggest that the syncytiotrophoblast is actively involved in the synthesis and metabolism of estrogens and in their role in placental endocrine function.

17-Hydroxysteroid Dehydrogenases↗

PubMed1987

Regulation of the synthesis of 3-hydroxy-3-methylglutaryl coenzyme A reductase in the bovine ovary in vivo and in vitro.

In order to investigate the pattern of ovarian cholesterol biosynthesis during the bovine estrous cycle, tissue concentrations of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, a rate-limiting enzyme in the synthesis of cholesterol, were determined by immunoblot techniques. Medium-sized (9-11 mm) and large (14-18 mm) follicles, after removal of follicular fluid by centrifugation, and corpora lutea from the early, early-mid, late-mid, and late stages of the luteal phase were used (n = 5 per group). The specific content (per microgram of tissue homogenate protein) and total content of HMG-CoA reductase in medium-sized and large follicles were substantially lower than those of corpora lutea of the early-mid and late-mid luteal phase. The specific content was elevated in a number of the corpora lutea from the early luteal phase and was low in regressing corpora lutea. Thus during the midluteal phase, when steroid hormone production is elevated, the total and specific contents of HMG-CoA reductase are also elevated. To investigate the mechanisms whereby the levels of HMG-CoA reductase are regulated, primary monolayer cultures of bovine luteal cells (early-mid and late-mid luteal phase) were used. Cells were cultured for 24 h in Dulbecco's modified Eagle's medium containing lipoprotein-poor fetal calf serum (2% vol/vol). At this concentration there was no stimulation of the production of progesterone above that seen with no addition of serum. Under these conditions the total and specific contents, and the synthesis, of HMG-CoA reductase were stimulated by treatment with (Bu)2cAMP (1 mM).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1987

Levels of messenger ribonucleic acid encoding cholesterol side-chain cleavage cytochrome P-450, 17 alpha-hydroxylase cytochrome P-450, adrenodoxin, and low density lipoprotein receptor in bovine follicles and corpora lutea throughout the ovarian cycle.

To investigate the molecular basis for the pattern of ovarian steroid production during the bovine estrous cycle, the relative levels of mRNA specific for cholesterol side-chain cleavage cytochrome P-450, 17 alpha-hydroxylase cytochrome P-450, adrenodoxin, and low density lipoprotein receptor were determined in ovarian antral follicles of differing size (less than 3-18 mm) and corpora lutea from the early, early-mid, late-mid, and regressionary stages. Total and poly(A)+ RNA was size-fractionated on agarose-formaldehyde gels, transferred to nylon filters and hybridized to specific 32P-labeled probes. The levels of mRNAs for the rate-limiting enzymes in the conversion of cholesterol into progesterone, namely cholesterol side-chain cleavage cytochrome P-450 and its electron donor, adrenodoxin, were higher in corpora lutea than in follicles. Conversely the levels of mRNA specific for the key regulatory enzyme in the conversion of pregnenolone or progesterone to androgen, namely 17 alpha-hydroxylase cytochrome P-450, were high in all antral follicles examined but were low in young corpora lutea and undetectable in more mature corpora lutea. Low density lipoprotein receptor mRNA was detectable in antral follicles and corpora lutea but the levels were greater in corpora lutea. These results suggest that the pattern of changes in steroid hormone biosynthesis during the bovine estrous cycle and in the ovarian content of steroidogenic enzymes is related to and probably dependent upon the pattern of change in levels of mRNAs for steroidogenic enzymes and related proteins.

Adrenodoxin↗

PubMed1987

Characterization of complementary deoxyribonucleic acid for human adrenocortical 17 alpha-hydroxylase: a probe for analysis of 17 alpha-hydroxylase deficiency.

To provide a basis for investigation of the molecular mechanisms underlying the hormonal regulation of steroid 17 alpha-hydroxylase (P-450 17 alpha) activity in adrenal, ovary, and testis as well as human 17 alpha-hydroxylase deficiency, we have isolated from a human fetal adrenal cDNA library a cDNA sequence complementary to the mRNA that encodes the human P-450 17 alpha enzyme. Of 75,000 colonies from the library that were screened by use of a nick-translated 5'-specific bovine P-450 17 alpha cDNA probe, 10 positive colonies were isolated and the clone with the longest insert (pcD-17 alpha H) was selected for further characterization. pcD-17 alpha H encodes the complete human P-450 17 alpha protein having approximately 78% homology at the nucleotide level and 71% homology at the amino acid level when the sequence of pcD-17 alpha H is compared to the bovine P-450 17 alpha cDNA sequence. By transient expression of the human P-450 17 alpha cDNA clone in COS 1 cells, we have demonstrated that the 17 alpha-hydroxylase and 17,20 lyase activities reside within the same human P-450 17 alpha polypeptide chain. The insert was also used as a probe to investigate, by means of Southern blot analysis, possible alterations in the P-450 17 alpha gene sequence in DNA isolated from skin fibroblasts from three patients with clinically characterized 17 alpha-hydroxylase deficiencies. No changes were detected in the DNA of any of the patients by this analysis.

Adrenal Hyperplasia, Congenital↗

PubMed1987

Regulation by follicle-stimulating hormone of the synthesis of aromatase cytochrome P-450 in human granulosa cells.

The effects of FSH to increase the activity of aromatase, as well as the synthesis of the components of the aromatase enzyme complex, have been studied in human ovarian granulosa cells obtained from women undergoing oocyte retrieval. FSH increased aromatase activity, as well as the synthesis of aromatase cytochrome P-450 (P-450AROM) in a time-dependent fashion, whereas in the absence of FSH, both activity and synthesis declined with duration of culture. The effect of FSH was mimicked by forskolin, an activator of adenylate cyclase. FSH also increased the synthesis of NADPH-cytochrome P-450 reductase, but to a relatively modest extent. The levels of hybridizable mRNA species encoding cytochrome P-450AROM of lengths 3.0, 2.4, and 1.6 kilobases were also increased with FSH treatment. It is concluded that the regulation of aromatase activity by FSH in human granulosa cells is mediated primarily by changes in the synthesis of cytochrome P-450AROM, that this action of FSH is mediated by cAMP, and that the changes in cytochrome P-450AROM synthesis are the consequences of changes in the levels of mRNA encoding this enzyme.

Aromatase↗

PubMed1987

Vasoactive intestinal peptide regulates cholesterol side-chain cleavage cytochrome P-450 (P-450scc) gene expression in granulosa cells from immature rat ovaries.

Vasoactive intestinal peptide (VIP), a neuropeptide present in ovarian nerves, has been previously shown to induce synthesis of the side-chain cleavage cytochrome P-450 enzyme which catalyzes the conversion of cholesterol to pregnenolone (the rate-limiting step in progesterone synthesis). In the present study we demonstrate, by means of a bovine 3'-specific P-450scc cDNA probe, that this VIP effect is exerted at least partially at the level of gene expression in cultured granulosa cells that were isolated from estrogen-primed, immature rats. The size and level of the 2.0 kilobase P-450scc mRNA species was assessed by Northern blot analysis, while the translatability of this mRNA was assayed by immunoisolation of the 35S-labeled P-450scc precursor protein translated from total RNA of control and stimulated granulosa cells. FSH was much more effective than VIP at increasing P-450scc mRNA concentrations in cultured granulosa cells, whereas secretin treatment was ineffective. The results suggest that, like FSH, the stimulatory effect of the neuropeptide VIP on ovarian progesterone secretion involves regulation of P-450scc gene expression during functional maturation of the prepubertal ovary.

Animals↗

PubMed1986

Expression of bovine 17 alpha-hydroxylase cytochrome P-450 cDNA in nonsteroidogenic (COS 1) cells.

Cortisol production requires the activity of only 17 alpha-hydroxylase, whereas the formation of sex steroids requires both 17 alpha-hydroxylase and 17,20-lyase activities. Studies in reconstituted enzyme systems have suggested that a single steroid hydroxylase, 17 alpha-hydroxylase cytochrome P-450 (P-450(17) alpha), catalyzes both activities. By expression of bovine adrenocortical P-450(17 alpha) in COS 1 (transformed monkey kidney) cells, which normally contain no detectable P-450(17) alpha, it has now been established in situ that a single polypeptide chain does catalyze both the 17 alpha-hydroxylase and the 17,20-lyase reactions. This heterologous system supports 17 alpha-hydroxylation of pregnenolone and progesterone with equal efficiency, but catalyzes about five times as much 17,20-lyase activity when 17 alpha-hydroxypregnenolone is the substrate than when 17 alpha-hydroxyprogesterone is the substrate. For these activities to be observed in COS 1 cells, newly synthesized apocytochrome P-450(17) alpha must bind heme and insert into the endoplasmic reticulum such that endogenous cytochrome P-450 reductase can support hydroxylation. Thus, COS 1 cells are a useful system for expression and study of various forms of cytochrome P-450.

Animals↗

PubMed1986

Bovine steroid 21-hydroxylase: regulation of biosynthesis.

A recombinant cDNA clone, PBC21-1, specific for bovine steroid 21-hydroxylase cytochrome P-450 (P-450C21) was identified in a bovine adrenocortical cDNA library, and this identity was confirmed by nucleotide sequencing which revealed significant amino acid homology (77%) with human P-450C21 cDNA. The pBC21-1 insert is 1.7 kilobases in length and includes a 1128 base pair region that encodes the C-terminal 376 amino acids of bovine P-450C21 as well as 535 base pairs of 3'-untranslated sequence. A novel feature of this insert is a 20 base pair intervening sequence near the 5' end, apparently the result of an aberrant splicing event. Northern blot analysis reveals that bovine P-450C21 is encoded by two transcripts, 2.3 and 2.0 kilobases in length which are detected in adrenal cortical RNA. Bovine liver, heart, kidney, and corpus luteum do not contain detectable P-450C21 transcripts. Regulation of P-450C21 gene expression by adrenocorticotropin was investigated with pBC21-1 and bovine adrenocortical cells in primary, monolayer culture. Treatment with ACTH or analogues of cAMP increases the steady-state levels of P-450C21 RNA in such cell cultures. In vitro transcription run-on assays suggest that this increase is, at least in part, due to the enhanced transcriptional activity of the P-450C21 gene.

Adrenal Cortex↗

PubMed1986

The insulin-like growth factor, somatomedin C, induces the synthesis of cholesterol side-chain cleavage cytochrome P-450 and adrenodoxin in ovarian cells.

The actions of insulin and somatomedin C (insulin-like growth factor I) on cholesterol side-chain cleavage activity and the synthesis of cytochrome P-450scc and adrenodoxin were investigated in primary cultures of swine ovarian (granulosa) cells. Nanomolar concentrations of pure human somatomedin C stimulated biosynthesis of progesterone and 20 alpha-hydroxypregn-4-en-3-one. Moreover, in the presence of exogenous sterol substrate for cholesterol side-chain cleavage, somatomedin C significantly enhanced pregnenolone biosynthesis in a time- and dose-dependent manner. This augmentation of functional cholesterol side-chain cleavage activity was accompanied by a dose-dependent (2-16-fold) increase in [35S]methionine incorporation into specific immunoprecipitable cytochrome P-450scc and adrenodoxin. Micromolar concentrations of insulin (but not proinsulin or desoctapeptide) also induced synthesis of cholesterol side-chain cleavage constituents by 4-7-fold. These results demonstrate that an insulin-like growth factor, somatomedin C, exerts discrete differentiating effects on ovarian cells characterized by increased synthesis of immunospecific cytochrome P-450scc and adrenodoxin. Thus, we infer that somatomedin C may serve a critical role in the differentiation of steroidogenic cells in the mammalian ovary.

Adrenodoxin↗

PubMed1986

Bovine adrenocortical cytochrome P-450(17 alpha). Regulation of gene expression by ACTH and elucidation of primary sequence.

In the steroidogenic pathway of the adrenal cortex, 17 alpha-hydroxylase cytochrome P-450 (P-450(17 alpha)) is a major regulatory enzyme. Previous studies have shown that stimulation of 17 alpha-hydroxylase activity occurs upon treatment of bovine adrenocortical cells in culture with adrenocorticotropic hormone (ACTH), via cAMP, due to an increase in the enzyme concentration. Alterations in the levels of this enzyme result in pronounced changes in the pattern of steroids produced, with the potent glucocorticoid cortisol, as well as the sex steroids, being products of 17 alpha-hydroxylated steroid precursors. In the present study, the identification and sequencing of two cloned DNA molecules, pB17 alpha-1 and pcD17 alpha-2, which are complementary to mRNA sequences encoding bovine adrenal cortex P-450(17 alpha), are reported. Clone pcD17 alpha-2 contains an open reading frame coding for the complete amino acid sequence of P-450(17 alpha) which consists of 509 amino acids and has a molecular weight of 57,251. By comparison to other forms of cytochrome P-450, we conclude that P-450(17 alpha) is a member of a previously unidentified family within the P-450 multigene superfamily. Single bovine and human mRNA species of approximately equal to 1850 bases in length hybridize to these clones. Regulation of the concentration of this RNA has been studied using bovine adrenocortical cells in culture. Treatment with ACTH or dibutyryl cAMP causes an increase in the content of P-450(17 alpha) RNA in as little as 2 h, and its concentration increases greater than 20-fold by 8 h. In contrast, other steroidogenic enzymes that have been studied respond more slowly to ACTH. Actinomycin D and cycloheximide block induction of P-450(17 alpha) RNA, indicating that regulation of 17 alpha-hydroxylase activity is controlled at the level of transcription and requires ongoing protein synthesis.

Adrenal Cortex↗

PubMed1986

Synergistic induction of monooxygenase activity by glucocorticoids and polycyclic aromatic hydrocarbons in human fetal hepatocytes in primary monolayer culture.

The ability of polycyclic aromatic hydrocarbons and glucocorticoids to regulate monooxygenase activity of human fetal liver has been studied using hepatocytes prepared by collagenase digestion of liver samples from human abortuses of 13 to 19 weeks of gestational age, and maintained in primary monolayer culture for periods up to 5 days. Addition of 1,2-benzanthracene to the cells caused an increase in monooxygenase activity (3-hydroxylation of benzo[a]pyrene and O-deethylation of 7-ethoxycoumarin) in a time-and concentration-dependent fashion. The concentration of 1,2-benzanthracene required to achieve half-maximal induction was 5 microM. The inductive effect of the polycyclic hydrocarbon was potentiated approximately 2.5-fold when dexamethasone (250 nM) or other glucocorticoids were included in the culture medium. Dexamethasone alone had little or no effect on the induction of monooxygenase activity. The concentration of dexamethasone required for half-maximal stimulation of monooxygenase activity in the presence of 1,2-benzanthracene was 5-10 nM, and the action of dexamethasone was reversed by the addition of cortisol 21-mesylate, consistent with the concept that the action of dexamethasone was mediated by binding to a glucocorticoid receptor. These results are suggestive that glucocorticoids, which are produced by the fetal adrenal and have an important role in the regulation of fetal development, act synergistically with polycyclic aromatic hydrocarbons to induce the activity of liver monooxygenases in the human fetus.

Aryl Hydrocarbon Hydroxylases↗

PubMed1986

Regulation of cytochrome P-450c by glucocorticoids and polycyclic aromatic hydrocarbons in cultured fetal rat hepatocytes.

The actions of polycyclic aromatic hydrocarbons and glucocorticoids to regulate the synthesis of cytochrome P-450c (the major isozyme induced by polycyclic aromatic hydrocarbons) were investigated in fetal rat hepatocytes maintained in primary monolayer culture. Treatment of hepatocytes in culture with 1,2-benzanthracene resulted in a 50-fold increase in 7-ethoxycoumarin O-deethylase activity. The level of P-450c increased in the cells in a time-dependent fashion as determined by immunoelectrophoretic analysis. The inductive effect of BA was potentiated approximately 1.6- to 2.3-fold when 1 microM dexamethasone was included in the culture medium. However, dexamethasone alone had little or no effect on the induction of P-450c. The rate of synthesis of P-450c was examined by immunoisolation of the specific isozyme from total cellular proteins radiolabeled with [35S]methionine and from the protein products formed during in vitro translation of the isolated mRNA. In addition, the amount of mRNA specific for cytochrome P-450c was determined by Northern blot analysis of RNA extracted from cultured cells. The changes in the rates of synthesis and mRNA levels were found to parallel the changes in enzyme activity. The concentration of dexamethasone required to cause a half-maximal increase in P-450c content in the presence of 1,2-benzanthracene was between 10(-8) and 10(-7) M. It is concluded that glucocorticoids act synergistically with polycyclic aromatic hydrocarbons to increase the levels of P-450c expressed in the fetal rat liver, and that this action is likely mediated by the classical type II glucocorticoid receptor.

Animals↗

PubMed1986

Regulation of cholesterol side-chain cleavage cytochrome P-450 gene expression in adrenal cells in monolayer culture.

Utilizing a cDNA probe specific for bovine cytochrome P-450scc and primary monolayer cultures of bovine adrenocortical cells and human fetal adrenal cells, it has been shown that the chronic action of ACTH on the adrenal cortex includes regulation of P-450scc gene expression at the transcriptional level. The bovine P-450scc cDNA hybridizes strongly to human, pig and rat RNA. Advantage was taken of the cross-reactivity of the bovine P-450scc cDNA with human P-450scc RNA to examine the regulation of P-450scc gene expression by ACTH in human fetal adrenal cells. This process is mediated by cyclic AMP and is inhibited by cycloheximide, in a fashion similar to bovine adrenocortical cells, suggestive that a protein factor(s) activates the response in both species. Hence, the actions of ACTH to regulate P-450scc gene expression in bovine adult adrenocortical cells and human fetal adrenal cells appear to proceed by similar mechanisms.

Adrenal Glands↗

PubMed1986

Transcriptional regulation of steroid hydroxylase genes by corticotropin.

Maintenance of optimal steroidogenic capacity in the adrenal cortex is the result of a cAMP-dependent response to the peptide hormone corticotropin (ACTH). The molecular mechanism of this action of ACTH has been examined by using five recombinant DNA clones specific for enzymes of the steroidogenic pathway (P-450scc, P-45011 beta, P-450C21, P-45017 alpha, and adrenodoxin). The presence of nuclear precursors in steady-state RNA samples derived from cultured bovine adrenocortical cells and moderate increases in the number of RNA chain initiations, as determined by in vitro nuclear run-off assays, indicate that ACTH controls the expression of the gene(s) for each of these proteins at the transcriptional level. The ACTH-mediated increase in accumulation of transcripts specific for steroid hydroxylases in nuclear RNA can be specifically blocked by inhibiting protein synthesis in bovine adrenocortical cell cultures. The steady-state concentrations of nuclear RNA for control genes show no decrease upon cycloheximide treatment. These studies suggest that a primary action of ACTH in the adrenal cortex is to activate (via cAMP) the synthesis of rapidly turning over protein factors that in turn mediate increased initiation of transcription of steroid hydroxylase genes. We propose that these protein factors impart specificity of induction to genes encoding components of this pathway in steroidogenic tissues.

Adrenal Cortex↗

PubMed1986

Isolation and characterization of a complementary DNA specific for human aromatase-system cytochrome P-450 mRNA.

A cloned complementary DNA sequence has been isolated from a human placental cDNA library in the bacteriophage expression vector lambda gt11 after screening with polyclonal antibodies against human placental aromatase-system cytochrome P-450 (P-450Arom). A single recombinant clone, lambda hAROM1, was characterized by its ability to generate a beta-galactosidase fusion protein that reacted independently with polyclonal antibodies raised against beta-galactosidase and cytochrome P-450Arom and with monoclonal antibodies specific for cytochrome P-450Arom. The cDNA insert, which was found to be 1.8 kilobases in length, was radiolabeled and used to analyze poly(A)+ RNA isolated from human placenta and total RNA isolated from human adipose stromal cells cultured in the absence or presence of regulatory factors. The radiolabeled cDNA hybridized to several size species of mRNA in both placental and adipose stromal cell RNA fractions. Changes in the levels of adipose stromal cell RNA that hybridized to the cDNA insert were associated with comparable changes in the levels of translatable cytochrome P-450Arom mRNA and aromatase system activity. These findings are indicative that lambda hAROM1 contains DNA sequences complementary to human cytochrome P-450Arom mRNA and are suggestive that regulatory factors affect aromatase activity by altering the transcriptional activity of the cytochrome P-450Arom gene.

Adipose Tissue↗

PubMed1986

Vasoactive intestinal peptide induces the synthesis of the cholesterol side-chain cleavage enzyme complex in cultured rat ovarian granulosa cells.

Vasoactive intestinal peptide (VIP) has been identified in ovarian nerves and stimulates steroid secretion from immature ovaries. To gain insight into its mechanism of action, the effect of VIP on the synthesis of the cholesterol side-chain cleavage enzyme complex was studied in ovarian granulosa cells from immature estrogen-primed rats. The cells were cultured for 48 hr in serum-free medium; the proteins were labeled with [35S]methionine; and the synthesis of cytochrome P-450, iron-sulfur protein, and NADPH:iron-sulfur protein reductase was evaluated by electrophoretic analysis after immunoisolation with polyclonal antibodies directed against the bovine adrenal enzymes. VIP at concentrations ranging from 0.001 to 1 microM stimulated 3- to 5-fold the synthesis of cytochrome P-450 and iron-sulfur protein. Peptide NH2-terminal histidine, COOH-terminal isoleucine, which has greater than 50% sequence homology of VIP, stimulated the synthesis of both proteins at approximately 50% of VIP effectiveness. Secretin, another member of the glucagon-secretin family of peptides, which has only 30% sequence homology to VIP, was without effect. Similar results were observed with the NADPH:iron-sulfur protein reductase. VIP-induced synthesis of the cholesterol side-chain cleavage enzyme complex was accompanied by a dose-related increase in cAMP accumulation and progestin formation. It is concluded that VIP regulates the synthesis of the ovarian cholesterol side-chain cleavage enzyme complex, which catalyzes the rate-limiting reaction in progesterone biosynthesis, and that the VIP effect is at least partially mediated through cAMP. It is suggested that a stimulatory action of VIP on the synthesis of ovarian progesterone may contribute to regulating the functional development of the ovary.

Animals↗

PubMed1986

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.

Adipose Tissue↗

PubMed1986

Cytochromes P-450scc, P-450(17)alpha, adrenodoxin, and reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase in bovine follicles and corpora lutea. Changes in specific contents during the ovarian cycle.

To investigate the basis for the pattern of ovarian steroid production during the bovine estrous cycle, the tissue concentrations of major steroidogenic enzymes, 17 alpha-hydroxylase cytochrome P-450 and cholesterol side-chain cleavage cytochrome P-450 (cytochrome P-450scc), and their respective electron donors, NADPH-cytochrome P-450 reductase and adrenodoxin, were estimated and compared with those of the nonsteroidogenic enzymes, cytochrome c oxidase and F1-ATPase. The levels of these enzymes were estimated in medium sized (9-11 mm) and large (14-18 mm) follicles after removal of follicular fluid by centrifugation, and corpora lutea from the early, early-mid late-mid, and late stages of the luteal phase (n = 5 per group). The specific contents of all enzymes and electron donors were determined by immunoblot analysis, except for cytochrome c oxidase, which was quantified by determination of specific activity. The specific (per microgram of tissue homogenate protein) and total (per follicle or corpus luteum) tissue contents of 17 alpha-hydroxylase cytochrome P-450 increased 0.5- and 5-fold respectively from medium sized to large follicles, but then decreased to undetectable levels in corpora lutea in the early luteal phase, and remained undetectable throughout the luteal phase. In contrast, the specific content of NADPH-cytochrome P-450 reductase was similar between follicles and corpora lutea. The specific contents of cytochrome P-450scc, adrenodoxin and cytochrome c oxidase in follicles were similar to those of corpora lutea of the early luteal phase. However, by the early-mid luteal phase the specific contents of luteal cytochrome P-450scc (490 +/- 46 vs. 5709 +/- 982 cpm/micrograms protein) and adrenodoxin (44 +/- 15 vs. 705 +/- 229 cpm/micrograms protein) were increased, by 12- and 15-fold, respectively (P less than 0.05). In contrast, cytochrome c oxidase activity (29.1 +/- 10.1 vs. 108.6 +/- 20.7 nmol/mg tissue protein X min) and the specific content of F1-ATPase increased only 3- to 4-fold reflective of an increase in numbers of mitochondria. The levels of these enzymes remained elevated until the late luteal phase when they declined markedly. It is concluded that the induction of synthesis of P-450scc and adrenodoxin after ovulation is specific and does not merely reflect biogenesis of mitochondria during luteinization. Moreover the changes in the types of steroids produced by the ovarian compartments throughout the estrous cycle are a reflection of changes in the tissue content of steroidogenic enzymes.

Adrenodoxin↗