Clascoterone for treatment of acne.

Clascoterone is a novel topical antiandrogen medication approved for the treatment of acne. Conventional oral antiandrogen treatments targeting acne such as combined oral contraceptives and spironolactone exert systemic hormonal effects which commonly preclude their usage in male patients while hampering their application in certain female patients. In contrast, clascoterone is a first-in-class antiandrogen proven to be both safe and effective for female and male patients above the age of 12. Outside of occasional localized skin irritation, clascoterone is usually well tolerated, however, some adolescents in a phase II clinical trial experienced biochemical evidence of HPA suppression, which resolved after discontinuing treatment. In this review, we provide an overview of clascoterone including its preclinical pharmacology, pharmacokinetics and metabolism, safety, clinical studies and indications.

Aldosterone and dexamethasone activate African lungfish mineralocorticoid receptor: Increased activation after removal of the amino-terminal domain.

Aldosterone, the main physiological mineralocorticoid in humans and other terrestrial vertebrates, first appears in lungfish, which are lobe-finned fish that are forerunners of terrestrial vertebrates. Aldosterone activation of the MR regulates internal homeostasis of water, sodium and potassium, which was critical in the conquest of land by vertebrates. We studied transcriptional activation of the slender African lungfish MR by aldosterone, other corticosteroids and progesterone and find that aldosterone, 11-deoxycorticosterone, 11-deoxycortisol and progesterone have half-maximal responses (EC50 s) below 1 nM and are potential physiological mineralocorticoids. In contrast, EC50 s for corticosterone and cortisol were 23 nM and 66 nM, respectively. Unexpectedly, truncated lungfish MR, consisting of the DNA-binding, hinge and steroid-binding domains, had a stronger response to corticosteroids and progesterone than full-length lungfish MR, indicating that the N-terminal domain represses steroid activation of lungfish MR, unlike human MR in which the N-terminal domain contains an activation function. BLAST searches of GenBank did not retrieve a GR ortholog, leading us to test dexamethasone and triamcinolone for activation of lungfish MR. At 10 nM, both synthetic glucocorticoids are about 4-fold stronger than 10 nM aldosterone in activating full-length lungfish MR, leading us to propose that lungfish MR also functions as a GR.

11-Deoxycortisol controls hydromineral balance in the most basal osmoregulating vertebrate, sea lamprey (Petromyzon marinus).

It is unknown whether and how osmoregulation is controlled by corticosteroid signaling in the phylogenetically basal vertebrate group Agnatha, including lampreys and hagfishes. It is known that a truncated steroid biosynthetic pathway in lampreys produces two predominant circulating corticosteroids, 11-deoxycortisol (S) and 11-deoxycorticosterone (DOC). Furthermore, lampreys express only a single, ancestral corticosteroid receptor (CR). Whether S and/or DOC interact with the CR to control osmoregulation in lampreys is still unknown. We examined the role of the endogenous corticosteroids in vivo and ex vivo in sea lamprey (Petromyzon marinus) during the critical metamorphic period during which sea lamprey increase osmoregulatory capacity and acquire seawater (SW) tolerance. We demonstrate in vivo that increases in circulating [S] and gill CR abundance are associated with increases in osmoregulatory capacity during metamorphosis. We further show that in vivo and ex vivo treatment with S increases activity and expression of gill active ion transporters and improves SW tolerance, and that only S (and not DOC) has regulatory control over active ion transport in the gills. Lastly, we show that the lamprey CR expresses an ancestral, spironolactone-as-agonist structural motif and that spironolactone treatment in vivo increases osmoregulatory capacity. Together, these results demonstrate that S is an osmoregulatory hormone in lamprey and that receptor-mediated discriminative corticosteroid regulation of hydromineral balance is an evolutionarily basal trait among vertebrates.

Cortexolone 17α-propionate (Clascoterone) Is a Novel Androgen Receptor Antagonist that Inhibits Production of Lipids and Inflammatory Cytokines from Sebocytes In Vitro

Cortexolone 17α-propionate (clascoterone) is a novel topical androgen antagonist that is being analyzed for its ability to treat acne. The pathogenesis of acne is attributed to multiple factors, including altered sebum production, inflammatory processes, dysregulation of the hormone microenvironment, and the proliferation of the skin commensal bacteria, Propionibacterium acnes (P. acnes). Androgens induce the proliferation and differentiation of sebocytes, (cells that comprise the sebaceous gland), help regulate the synthesis of the lipids that are incorporated into sebum and stimulate the production of cytokines that are found in inflammatory acne lesions. Several studies have established that clascoterone is a potent antiandrogen that is well tolerated and has selective topical activity. Its potency as an acne therapeutic is currently being analyzed in a large phase 3 clinical trial. The study described herein elucidates for the first time the mechanism of action of clascoterone. Clascoterone was found to bind the androgen receptor (AR) with high affinity in vitro, inhibit AR-regulated transcription in a reporter cell line, and antagonize androgen-regulated lipid and inflammatory cytokine production in a dose-dependent manner in human primary sebocytes. In particular, when compared to another AR antagonist, spironolactone, clascoterone was significantly better at inhibiting inflammatory cytokine synthesis from sebocytes. Therefore, clascoterone may be an excellent candidate to be the first topical antiandrogen to treat acne. J Drugs Dermatol. 2019;18(5):412-418.

Cortexolone 17α-Propionate (Clascoterone) is an Androgen Receptor Antagonist in Dermal Papilla Cells In Vitro

Cortexolone 17α-propionate (clascoterone) is a novel androgen antagonist that is currently being analyzed in a large phase 2 clinical trial for the topical treatment of androgenetic alopecia (AGA). While the pathogenesis of AGA is still debated, the consensus is that AGA is an androgen-dependent hair disorder with strong genetic links, and that the testosterone metabolite, dihydrotestosterone (DHT), plays a causal role in its development. DHT binds to the androgen receptor (AR) in scalp dermal papilla cells (DPC) to induce AR-mediated transcription of genes that contribute to AGA in genetically predisposed individuals. Several studies have established that clascoterone is a potent antiandrogen that is well tolerated and has selective topical activity. The study described herein elucidates a potential mechanism of clascoterone in AGA. Clascoterone was found to inhibit AR-regulated transcription in a reporter cell line with similar efficacy to the 5α-reductase inhibitor, finasteride. More importantly, when compared with another direct AR antagonist, enzalutamide, clascoterone was significantly better at inhibiting IL-6 synthesis from DHT-stimulated primary cultures of human scalp DPC. Therefore, clascoterone may be an excellent candidate to be the first topical antiandrogen for treating AGA. J Drugs Dermatol. 2019;18(2):197-201.

Corticosteroid and progesterone transactivation of mineralocorticoid receptors from Amur sturgeon and tropical gar.

The response to a panel of steroids by the mineralocorticoid receptor (MR) from Amur sturgeon and tropical gar, two basal ray-finned fish, expressed in HEK293 cells was investigated. Half-maximal responses (EC50s) for transcriptional activation of sturgeon MR by 11-deoxycorticosterone, corticosterone, 11-deoxycortisol, cortisol and aldosterone, and progesterone (Prog) were between 13 and 150 pM. For gar MR, EC50s were between 8 and 55 pM. Such low EC50s support physiological regulation by these steroids of the MR in sturgeon and gar. Companion studies with human and zebrafish MRs found higher EC50s compared with EC50s for sturgeon and gar MRs, with EC50s for zebrafish MR closer to gar and sturgeon MRs than was human MR. For zebrafish MR, EC50s were between 75 and 740 pM; for human MR, EC50s were between 65 pM and 2 nM. In addition to Prog, spironolactone (spiron) and 19nor-progesterone (19norP) were agonists for all three fish MRs, in contrast with their antagonist activity for human MR, which is hypothesized to involve serine-810 in human MR because all three steroids are agonists for a mutant human Ser810Leu-MR. Paradoxically, sturgeon, gar, and zebrafish MRs contain a serine corresponding to serine-810 in human MR. Our data suggest alternative mechanism(s) for Prog, spiron, and 19norP as MR agonists in these three ray-finned fishes and the need for caution in applying data for Prog signaling in zebrafish to human physiology.

Involvement of epidermal growth factor receptors and mitogen-activated protein kinase in progestin-induction of sperm hypermotility in Atlantic croaker through membrane progestin receptor-alpha.

The intracellular pathways mediating rapid, nongenomic progestin stimulation of sperm motility remain unclear. The role of epidermal growth factor receptors (Egfr and ErbB2) and mitogen-activated protein kinase (Mapk) in membrane progestin receptor-alpha (mPRα)-mediated progestin stimulation of sperm hypermotility was examined in a teleost, Atlantic croaker. Inhibition of upstream regulators of Egfr, intracellular tyrosine kinase (Src) with PP2, and matrix metalloproteinase (MMP) with Ilomastat, abolished progestin-initiated sperm hypermotility by 17,20ß,21-trihydroxy-4-pregnen-3-one (20ß-S; 20 nM) and a specific mPRα agonist, Org OD 02-0 (20 nM). Pretreatment of croaker sperm with EGFR inhibitors, AG1478 (5 µM) and RG13022 (50 µM), the ErbB2 inhibitor, AG879 (5 nM), or the MEK1/2 inhibitor, U0126 (500 nM) blocked progestin stimulation of sperm motility. Levels of phosphorylated extracellular-related kinase 1 and 2 (P-Erk1/2) were increased after 20ß-S treatment. These results demonstrate that progestin-mediated hypermotility via mPRα in croaker sperm involves activation of the Egfr, ErbB2 and Mapk pathways.

AroER tri-screen™ is a novel functional assay to estimate both estrogenic and estrogen precursor activity of chemicals or biological specimens.

The purpose of the study is to define AroER tri-screen's utility for identifying endocrine-disrupting chemicals (EDCs) that target aromatase and/or estrogen receptor (ER), and to measure the total estrogenic activity in biological specimens. ER-positive, aromatase-expressing MCF-7 breast cancer cells were stably transfected with an estrogen responsive element (ERE)-driven luciferase reporter plasmid to yield a new high-throughput screening platform-the AroER tri-screen. AroER tri-screen was capable of identifying estrogen precursors, such as cortodoxone, which function as estrogens through a two-step conversion process in aromatase-expressing tissue. Furthermore, the system proved useful for assessing EDC activity in biologically relevant samples. Estimating these activities is critical because natural estrogens and estrogenic EDCs are important factors in ER-positive breast cancer risk. As our research demonstrates, incorporating functionally active aromatase into the AroER tri-screen produces a powerful and unique tool to (1) identify new EDCs targeting aromatase and/or ER; (2) discover novel EDCs activated by aromatase; and (3) estimate overall estrogenic activities in biological samples as a potential intermediate risk factor for breast cancer.

Membrane progestin receptor alpha mediates progestin-induced sperm hypermotility and increased fertilization success in southern flounder (Paralichthys lethostigma).

Progestin hormones stimulate sperm motility in teleosts but their mechanisms of action remain unclear. Preliminary results suggest that progestin upregulation of sperm motility in southern flounder and several other marine species is mediated through a sperm membrane progestin receptor with the characteristics of membrane progestin receptor alpha (mPRα, also known as Paqr7b). The hypothesis that mPRα has an important role in progestin regulation of southern flounder sperm motility and fertility was tested in the present study. The specific mPRα agonist, 10-ethenyl-19-norprogesterone (Org OD 02-0, 100nM), mimicked the stimulatory actions of the endogenous progestin, 17,20ß, 21-trihydroxy-4-pregnen-3-one (20ß-S, 100nM) on flounder sperm motility. The concentration of the mPRα protein on sperm plasma membranes was positively correlated to sperm motility as well as the responsiveness of sperm to progestin stimulation. Acute in vitro progestin treatment of sperm with high mPRα protein levels increased both sperm motility and fertilization success in strip spawning experiments. However, in vitro progestin treatments were ineffective on sperm with low receptor abundance. A single injection of the superactive gonadotropin-releasing hormone analog (LHRHa, 100µg/kg) increased sperm motility and fertilization success in strip spawning experiments 72h post-injection which was accompanied by an increase in mPRα protein concentrations on sperm plasma membranes. These results provide clear evidence that southern flounder sperm hypermotility is mediated through mPRα. Stimulatory G proteins, but not inhibitory G proteins, were identified in flounder sperm plasma membrane fractions. The finding that treatment of flounder sperm plasma membrane fractions with either 20ß-S or Org OD 02-0 increases cAMP levels suggests progestins stimulate flounder sperm motility by activating an mPRα/stimulatory G protein/membrane adenylyl cyclase pathway. A similar mechanism has been identified in Atlantic croaker, suggesting that the signaling pathway mediated by mPRα in sperm is highly conserved in advanced teleosts. Collectively, our results indicate that progestin-stimulation of flounder sperm hypermotility and fertility is dependent on a sufficient concentration of mPRα which can be upregulated by in vivo LHRHa treatments. These findings potentially have practical applications for enhancing the fertility of male flounder broodstock.

11-Deoxycortisol impedes GABAergic neurotransmission and induces drug-resistant status epilepticus in mice.

Systemic injection of high doses of 11-deoxycortisol succinate had been reported to induce status epilepticus in rats and cats that was associated with paroxysmal epileptiform activity refractory to first generation antiepileptic drugs (AEDs). Using patch clamp recordings we have investigated the mechanisms of 11-deoxycortisol-induced excitability and we have discovered that this molecule accelerates the decay time of the inhibitory postsynaptic currents (IPSCs) mediated by GABA(A) receptors, both in neuronal cultures and in hippocampal slices. In addition, it reduces the amplitude and frequency of IPSCs. Thus, 11-deoxycortisol action on GABAergic neurotransmission may be one of the underlying causes of convulsive seizures that had been observed in rats. In the present study, we have reproduced the ability of 11-deoxycortisol to induce convulsive seizures after intravenous infusion in mice. The threshold dose of 11-deoxycortisol necessary for seizure induction was also determined (0.95 mmol/kg). Furthermore, we have established that these seizures are completely refractory to several AEDs such as phenytoin (up to 100 mg/kg), carbamazepine (up to 56 mg/kg), and valproate (up to 300 mg/kg). Levetiracetam and diazepam afforded only limited protection at high doses, 540 and 3-10 mg/kg, respectively. Interestingly, long-lasting seizures induced by 11-deoxycortisol in mice were not associated with typical neuropathological changes observed in other models of status epilepticus. We propose that 11-deoxycortisol-induced seizures may be an advantageous experimental model of drug-resistant epilepsy. Finally, better understanding of the pro-epileptic properties of 11-deoxycortisol is very important, because this endogenous steroid precursor may play a role in the pathophysiology of epilepsy. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.

Identification of 17,20ß,21-trihydroxy-4-pregnen-3-one (20ß-S) receptor binding and membrane progestin receptor alpha on southern flounder sperm (Paralichthys lethostigma) and their likely role in 20ß-S stimulation of sperm hypermotility.

The existence of direct progestin actions on teleost sperm to stimulate hypermotility is not widely acknowledged because it has only been demonstrated in members of the family Sciaenidae. In the present study, progestin stimulation of sperm hypermotility was investigated in a non-sciaenid, southern flounder, and the potential role of membrane progestin receptor alpha (mPRα or Paqr7b) in mediating this action was examined. The major progestin produced in vitro by flounder testicular fragments co-migrated with 17,20ß,21-trihydroxy-4-pregnen-3-one (20ß-S) during thin-layer chromatography. Treatment of flounder sperm with 5 nM-100 nM 20ß-S significantly increased sperm velocity in vitro, whereas 17,20ß-dihydroxy-4-pregnen-3-one and other steroids were ineffective. A single class of high affinity (K(d) 22.95 nM), saturable, limited-capacity binding sites (B(max) 0.013 nM) specific for 20ß-S was identified on sperm membranes. Treatment of sperm membranes with guanosine 5'-(3-O-thio)triphosphate reduced [(3)H]-20ß-S binding, suggesting the 20ß-S receptor couples to a G protein. The membrane adenylyl cyclase inhibitor 2',5'-dideoxyadenosine blocked 20ß-S-induced sperm hypermotility, indicating 20ß-S activates stimulatory G proteins. Finally, flounder paqr7b was cloned and characterized from testicular tissues. The Paqr7b protein is expressed on the midpiece of flounder sperm and is more abundant in individuals with high sperm motility than low motility donors. These findings suggest that 20ß-S stimulates sperm hypermotility in flounder through activation of stimulatory G proteins, likely through Paqr7b. The finding that progestins directly stimulate sperm hypermotility in a flatfish, a highly derived species not belonging to the teleost family Sciaenidae, suggests this phenomenon is widespread among advanced fishes.

Mineralocorticoids restore quiescent morphology and reduce VEGF receptor expression in inflamed choroidal endothelial cells in vitro.

BACKGROUND/AIMS: While the glucocorticoid triamcinolone acetonide (9alpha-fluoro-16alpha-hydroxyprednisolone, TA) has been widely administered as a treatment of ocular inflammation, mineralocorticoids have not been tested for their efficacy. METHODS: We assessed cellular morphology and actin distribution by immunomicroscopy and light microscopy, membrane permeability with transendothelial resistance and cell surface vascular endothelial growth factor receptor-1 (VEGF-R1) expression by flow cytometry. RESULTS: Fludrocortisone acetate was more effective than TA in restoring quiescent morphology and reducing membrane permeability in phorbol-12-myristate-acetate (PMA)-stimulated choroidal endothelial cells (CECs). Each of the corticosteroids inhibited VEGF-R1 cell surface expression in PMA-responsive CECs. CONCLUSION: Mineralocorticoids may be of potential use in reducing vascular permeability in ocular disease.

Biotransformation of cortexolone to hydrocortisone by molds using a rapid color development assay.

Capabilities of 22 molds were assessed for 11beta-hydroxylation of cortexolone (Reichstein's compound S) to hydrocortisone. The biotransformation capability was compared for solid-state and submerged monocultures of the molds under otherwise identical conditions. A novel rapid color development assay and thin layer chromatography were used to qualitatively establish the ability of the fungi to convert cortexolone to hydrocortisone. These assays were validated and supplemented with data from high performance liquid chromatography to obtain quantitative information on the biotransformation. Nearly all the fungi consumed a significant fraction of the cortexolone fed, but only four (i.e. two isolates of Cunninghamella blakesleeana, C. echinulata and Curvularia lunata) yielded measurable quantities of hydrocortisone. Submerged cultures generally gave significantly greater yield of hydrocortisone compared to equivalent solid-state cultures.

Immunoenzymometric assay for a small molecule,11-deoxycortisol, with attomole-range sensitivity employing an scFv-enzyme fusion protein and anti-idiotype antibodies.

To overcome the sensitivity limit in immunoassays for small molecules (haptens), we established a noncompetitive immunoenzymometric assay (IEMA) format that can detect attomole-range hapten molecules. We selected 11-deoxycortisol (11-DC; Mr 346.5), a corticosteroid serving a diagnostic index for pituitary-adrenal function, as a model target hapten. A fusion of a single-chain Fv fragment (scFv) specific for 11-DC and alkaline phosphatase (ALP) was generated for use as an enzyme-labeled antibody, instead of the conventional chemically linked enzyme-antibody conjugates. After binding reaction of 11-DC and fixed amounts of the fusion protein (scFv-ALP), the unbound fusion protein was removed by incubation with a mouse beta-type anti-idiotype antibody recognizing the scFv paratope. These complexes were captured by magnetic separation using anti-mouse IgG antibody-coated magnetic beads. Following magnetic sedimentation of the beads, immune complexes of scFv-ALP and 11-DC remained in the supernatant were further purified by capture on microtiter plates with immobilized alpha-type anti-idiotype antibody. As measured fluorometrically, ALP activity from bound immune complexes on the plates increased with increasing 11-DC, which is characteristic of a noncompetitive relationship. This IEMA afforded an extremely low detection limit (20 amol/assay), a very wide measurable range, and practical specificity. The plasma 11-DC levels determined for healthy subjects were validated as reliable.

The effect of the corticosteroid hormone cortexolone on the metabolites produced during phenanthrene biotransformation in Cunninghamella elegans.

The metabolism of phenanthrene and the mammalian corticosteroid hormone cortexolone by the fungus Cunninghamella elegans was studied. The amounts of the cortexolone transformation products, cortisol and epicortisol, were affected by the presence of phenanthrene. Approximately 40% more cortisol was produced by C. elegans in cultures with phenanthrene. In contrast, epicortisol formation decreased. C. elegans transformed phenanthrene to phenanthrene trans-1,2-,3,4-, and 9,10-dihydrodiols, phenols, diphenols (diols) and glucoside conjugates of 1-, 2-, 3-, 4-, and 9-phenanthrols. Almost all of the phenanthrene initially added was metabolized to ethyl acetate extractable metabolites. In the mycelia and culture medium extracts, phenanthrol glucosides represented 80% and 94% of the total metabolites, respectively. The major metabolite was the glucoside conjugate of 1-phenanthrol. The presence of cortexolone affected the biodegradation of phenanthrene by decreasing the amounts of phenanthrene metabolites compared to control cultures.

In the ventral tegmental area, progestins' membrane-mediated actions for lordosis of hamsters and rats involve protein kinase A.

Progestin-facilitated lordosis of hamsters and rats is enhanced by activation of dopamine type 1 (D1) or GABAA/benzodiazepine receptor complexes (GBRs) in the ventral tegmental area (VTA) and these effects involve G-proteins and second messengers, such as adenosine 3',5'-monophosphate (cAMP). We examined whether D1- and/or GBR-mediated increases in progestin-facilitated lordosis of female hamsters and rats involve the cAMP-dependent protein kinase, protein kinase A (PKA), in the VTA. In experiment 1, ovariectomized hamsters, primed with estradiol (E2; 10 microg at h 0) + progesterone (P; 100 microg at h 45), were first pre-tested for lordosis and motor behavior (h 48) and then infused with the PKA inhibitor, Rp-cAMP (100 ng/side), or vehicle. Thirty minutes later, hamsters were retested and then received infusions of the D1 agonist, SKF38393 (100 ng/side), the GBR agonist, muscimol (100 ng/side), or vehicle to the VTA. Hamsters were post-tested for lordosis and motor behavior 30 min later. In Experiment 2, ovariectomized rats, primed with E2 (10 microg at h 0), were first pre-tested for lordosis and then infused with Rp-cAMP (100 ng/side) or vehicle to the VTA at h 44. Immediately after testing, rats received infusions of SKF38393 (100 ng/side), muscimol (100 ng/side), or vehicle and were retested for lordosis. Rats were then infused with the neurosteroid, 5alpha-pregnan-3alpha-ol-20-one (3alpha,5alpha-THP; 100 or 200 ng/side), or beta-cyclodextrin vehicle and were post-tested for lordosis and motor behavior 10 and 60 min later. The enhancing effects of progestins or progestins plus D1 or GBR activation on lordosis of E2-primed hamsters and rats were blocked by the PKA inhibitor, Rp-cAMP. Thus, in the VTA, progestins' membrane actions involving D1 or GBRs are mediated, in part, by PKA.

Pharmacological profile of 9,11-dehydrocortexolone 17alpha-butyrate (CB-03-04), a new androgen antagonist with antigonadotropic activity.

A series of new cortexolone-related derivatives has been synthesized and investigated for potential anti-androgenic activity. Among the steroids evaluated, 9,11-dehydrocortexolone 17alpha-butyrate (CB-03-04) was the most promising one. The compound displayed a strong local antiandrogenic activity in hamster's flank organ test, and it was also found to be effective in the rat after subcutaneous injection. When compared to other well known androgen antagonists, the rank order of topical anti-androgenic activity in that test was: cyproterone acetate (CAS 427-51-0) > or = CB-03-04 > finasteride (CAS 98319-26-7) > flutamide (CAS 13311-84-7). In addition, the steroid had selective antigonadotropic activity, when injected into parabiotic rats, and was about as active as progesterone. The activity of CB-03-04 was ascribed mainly to its ability to compete with the stimulating effects of testosterone and dihydrotestosterone and, concurrently, to inhibit the gonadotropins hypersecretion. This bimodal mechanism of action could be predictive for the clinical usefulness of the steroid in the treatment of prostate cancer and benign prostate hypertrophy.

Activation of a pertussis toxin-sensitive, inhibitory G-protein is necessary for steroid-mediated oocyte maturation in spotted seatrout.

Oocyte maturation (OM) is initiated in lower vertebrates and echinoderms when maturation-inducing substances (MIS) bind oocyte membrane receptors. This study tested the hypothesis that activation of a G(i) protein is necessary for MIS-mediated OM in spotted seatrout. Addition of MIS significantly decreased adenylyl cyclase activity in a steroid specific, pertussis toxin (PTX)-sensitive manner in oocyte membranes and microinjection of PTX into oocytes inhibited MIS-induced OM, suggesting the steroid activates a G(i) protein. MIS significantly increased [(35)S]GTPgammaS binding to ovarian membranes, confirming that MIS receptor binding activates a G-protein, and immunoprecipitation studies showed the increased [(35)S]GTPgammaS binding was associated with Galpha(i1-3) proteins. Radioligand binding studies in ovarian membranes using GTPgammaS and PTX demonstrated that the MIS binds a receptor coupled to a PTX-sensitive G-protein. This study provides the first direct evidence in a vertebrate model that MIS-induced activation of a G(i) protein is necessary for OM. These results support a mechanism of MIS action involving binding to a novel, G-protein coupled receptor and activation of an inhibitory G-protein, the most comprehensive and plausible model of MIS initiation of OM proposed to date.

Steroid-induced oocyte maturation in Atlantic croaker (Micropogonias undulatus) is dependent on activation of the phosphatidylinositol 3-kinase/Akt signal transduction pathway.

Exposure of fully grown fish and amphibian oocytes to a maturation-inducing steroid (MIS) activates numerous signal transduction pathways to initiate the final stage of oocyte maturation. These events culminate in the activation of maturation-promoting factor and germinal vesicle breakdown (GVBD). In most species, exposure to MIS causes a transient decrease in oocyte cAMP levels. Whether this reduction in oocyte cAMP concentration is sufficient to induce GVBD is unclear. The current study tested the hypothesis that activation of cAMP-independent signal transduction pathways by the naturally occurring MIS, 17,20beta,21-trihydroxy-4-pregnen-3-one (20beta-S), is necessary for GVBD in Atlantic croaker (Micropogonias undulatus) oocytes. Results indicate that although 20beta-S treatment of oocyte membranes significantly reduced cAMP production, incubation of follicles with the cell-permeable cAMP-dependent protein kinase (Prka) inhibitors Rp-cAMP or KT5720 did not promote GVBD in the absence of 20beta-S. Additionally, treatment of follicles with the phosphodiesterase (Pde) inhibitors Cilostamide (Pde3) or Rolipram (Pde4) significantly reduced GVBD, but they were not able to completely block it. In contrast, pharmacologic inhibition of the cAMP-independent phosphatidylinositol 3-kinase (Pik3)/Akt signal transduction pathway using the Pik3 inhibitors Wortmannin or LY294002, or the Akt inhibitor ML-9, blocked 20beta-S-induced GVBD. Finally, mitogen-activated protein kinase (Mapk1/3) activity increased after treatment with 20beta-S; however, inhibition of Mapk1/3 activity using PD98059 or U0126 had no effect on GVBD. These results demonstrate that activation of cAMP-independent signaling pathways, especially the Pik3/Akt pathway, is necessary for 20beta-S-induced GVBD in Atlantic croaker oocytes.