Abstract Zinc is an essential trace element involved in numerous biological processes, including cellular signalling, development, and reproduction. Zinc homeostasis is regulated by zinc transporters, yet the physiological roles of many transporters remain poorly understood in vivo . Here, we investigated the function of the zinc transporter ZIP9 (SLC39A9) using a zebrafish ( Danio rerio ) knockout model. Elemental imaging using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) revealed altered zinc distribution in zip9 -deficient larvae. Synchrotron-based X-ray fluorescence (XRF) imaging further showed reduced zinc levels in the brain region of mutant zebrafish. Consistent with these observations, loss of zip9 was associated with altered expression of key neuroendocrine genes within the hypothalamic-pituitary-gonadal (HPG) axis. Zip9 mutant females exhibited disrupted ovarian follicle development, reduced spawning rates, and decreased egg production. In addition, embryos derived from zip9 mutant parents displayed reduced size, impaired early development, and decreased survival. Together, these findings identify ZIP9 as a regulator of zinc distribution in vivo and suggest that ZIP9-mediated zinc signalling contributes to reproductive regulation in zebrafish.

The classic genomic mechanism of steroid action involves activation of nuclear receptors and their translocation to the nucleus where they alter gene transcription. However, steroids also exert a wide variety of nongenomic actions initiated at or near the cell surface through novel membrane receptors and nuclear steroid receptors and in other extranuclear locations resulting in rapid activation of intracellular signaling pathways. Recent progress has been made in identifying rapid androgen actions and the receptors that mediate them in the male reproductive system. In this article, the signaling pathways and receptors involved in nongenomic androgen actions in the male reproductive tissues are briefly reviewed.

Sub-optimal milt production and sperm quality are major causes of relatively poor fertilization and reproductive success of southern flounder (flounder, Paralichthys lethostigma) in captivity. Previous work showed that progestin improved low motility of sperm and increased fertility of male flounder through membrane progestin receptor-mediated Egf/epidermal growth factor receptor (Egfr) signaling and that this effect can be mimicked by acute in vitro treatments with recombinant human Egf. However, knowledge of seasonal changes in southern flounder sperm Egf/Egfr physiology and confirmation that homologous recombinant southern flounder Egf exerts similar effects on flounder sperm is required to develop practical aquaculture methods to increase male broodstock fertility with recombinant Egf. Here, southern flounder Egf and Egfr were cloned and shown by phylogenetic analysis to have extensive sequence homologies with those of Japanese flounder and several other flatfish species. Egf and Egfr mRNAs and proteins were detected in southern flounder testes and sperm by RT-PCR and Western blotting with specific antibodies, respectively. There was a significant decrease in Egf mRNA expression in milt towards the end of the spawning season whereas Egfr mRNA and protein expression were unchanged. Recombinant southern flounder Egf, generated in a yeast expression system, was highly effective in increasing sperm motility and fertilization success. The results suggest that when endogenous Egf levels have decreased towards the end of the spawning season, Egfr on flounder sperm can still be activated with recombinant flounder Egf treatments to enhance the reproductive performance of male southern flounder broodstock in aquaculture farms.

Development of southern flounder commercial aquaculture is limited by poor sperm motility and fertility, but the underlying causes and mechanisms regulating sperm motility are unknown. In vitro treatment of southern flounder sperm with progestin hormones rapidly increases velocity (swimming speed), a major component of sperm motility, and also fertility through membrane progestin receptor alpha (mPR alpha) and increases in Acy (adenylyl cyclase)/cAMP signaling. The progestin-induced increase of Atlantic croaker sperm velocity is also mediated through epidermal growth factor receptor (Egfr) transactivation resulting in extracellular-regulated kinase 1/2 (Erk1/2) signaling, but its role in regulating southern flounder sperm velocity is unknown. The hypotheses that Egfr transactivation and Erk1/2 signaling mediates progestin-induced increases in sperm velocity and calcium levels in southern flounder, and that direct stimulation of sperm Egfr/Erk1/2 and Acy/cAMP pathways increases sperm fertility in strip spawning trials were tested in the present study. Golf, Egfr, Erk1/2, and protein kinase A were detected on southern flounder sperm by immunocytochemistry. Involvement of Egfr/Erk1/ 2 signaling in increasing sperm velocity and calcium levels was investigated by preincubating sperm with two EGFR inhibitors, AG1478, and AG825, a matrix metalloproteinase inhibitor, Ilomastat, and two ERK1/2 inhibitors, PD98059, and U0126, for 30 min prior to 1 min treatment with progestins. Sperm swimming speed was observed under a microscope after activation with a hyperosmotic medium and recorded for 1 min. Whereas treatment with the inhibitors did not alter basal sperm velocity and calcium levels, all of them significantly attenuated velocity and calcium increases in response to progestins. Treatment in vitro for 1 min with the Egfr agonist, recombinant human EGF (0.1 nM and 100 nM), mimicked the stimulatory effects of progestins on sperm velocity, the increase in calcium, and also fertility in a strip spawning trials with ovulated southern flounder eggs. The results suggest Egfr/Erk1/2 signaling is involved in sperm velocity and fertility responses to progestins in southern flounder. Direct in vitro treatment with the Acy activator, forskolin (10 mu M) for 1 min mimicked the stimulatory action of progestins on sperm calcium levels, velocity, and fertility, suggesting that Acy/cAMP signaling also mediates increased sperm swimming speed and fertility. Importantly, both EGF and forskolin also directly stimulated southern flounder sperm velocity and fertility at the end of the reproductive season when sperm were no longer responsive to progestins. The results suggest that EGF and forskolin could potentially be used as pharmacological agents to enhance reproductive performance of male southern flounder broodstock.

In the original publication [...].

Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide, and current treatment options are unsatisfactory on the long term. Several studies suggest a potential neuroprotective action by female hormones, especially estrogens. The potential role of progestogens, however, is less defined, and no studies have investigated the potential involvement of membrane progesterone receptors (mPRs). In the present study, the putative neuroprotective role for mPRs was investigated in SH-SY5Y cells, using two established pharmacological treatments for cellular PD models, 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpyridinium (MPP+). Our results show that both the physiologic agonist progesterone and the specific mPR agonist Org OD 02-0 were effective in reducing SH-SY5Y cell death induced by 6-OHDA and MPP+, whereas the nuclear PR agonist promegestone (R5020) and the GABAA receptor agonist muscimol were ineffective. Experiments performed with gene silencing technology and selective pharmacological agonists showed that mPRα is the isoform responsible for the neuroprotective effects we observed. Further experiments showed that the PI3K-AKT and MAP kinase signaling pathways are involved in the mPRα-mediated progestogen neuroprotective action in SH-SY5Y cells. These findings suggest that mPRα could play a neuroprotective role in PD pathology and may be a promising target for the development of therapeutic strategies for PD prevention or management.

Substantial progress has been made in our understanding of the nongenomic actions, ligand binding, intracellular signaling pathways, and functions of membrane progesterone receptors (mPRs) in reproductive and nonreproductive tissues since their discovery 20 years ago. The five mPRs are members of the progestin adipoQ receptor (PAQR) family which also includes adiponectin receptors (AdipoRs). However, unlike AdipoRs, the 3-D structures of mPRs are unknown, and their structural characteristics remain poorly understood. The mechanisms regulating mPR functions and their trafficking to the cell surface have received little attention and have not been systematically reviewed. This paper summarizes some structural aspects of mPRs, including the ligand binding pocket of mPRα recently derived from homology modeling with AdipoRs, and the proposed topology of mPRs from the preponderance of positively charged amino acid residues in their intracellular domains. The mechanisms of trafficking membrane receptors to the cell surface are discussed, including the amino acid motifs involved with their export to the cell surface, the roles of adaptor proteins, and post-translational glycosylation and palmitoylation modifications that promote cell surface expression and retention. Evidence for similar mechanisms regulating the expression and functions of mPRs on the cell surface is discussed, including the identification of potential export motifs on mPRα required for its trafficking to the cell membrane. Collectively, these results have identified several potential mechanisms regulating the expression and functions of mPRs on the cell membrane for further investigation.

Abstract Disclosure: P. Thomas: None. The zinc transporter ZIP9 also functions as a membrane androgen receptor (mAR) that mediates androgen-dependent rapid signaling and pro- or anti-apoptotic, proliferative, and migratory responses in fish, rodent, and human cells. Testosterone activation of ZIP9 in two triple-negative breast cancer cell lines, MDA-MB-468 cells and MDA-MB-231 cells transfected with ZIP9 (231-ZIP9), induces G-protein-dependent second messenger signaling and increases in intracellular free zinc. However, the relative importance of these two signaling pathways in the regulation of pro- and anti-tumorigenic responses in these cell lines is unclear because both androgen signaling and zinc status influence tumorigenesis in breast tissues. The effects of overexpression of ZIP9 on free zinc status, G protein- and zinc-dependent regulation of migration, and migration marker expression in 231-ZIP9 cells in the absence of testosterone were compared to those observed in MDA-MB-231 cells transfected with vector alone (231-vect). Migration and invasion of 231-ZIP9 cells were significantly increased after 24-48 hr culture in serum-free media compared to 231-vect cells which was accompanied by a significant increase in free zinc levels. Treatment of 231-vect cells with a zinc ionophore also increased migration, whereas migration of 231-ZIP9 cells was attenuated by treatment with an intracellular zinc chelator, TPEN. Expression of the migration markers, MYL9 and CYR61, was increased in 231-ZIP9 after 24-48 hr culture, whereas expression of apoptotic markers was decreased compared to 231-vect controls. Proximity Ligation Analysis revealed that ZIP9 is closely associated with a stimulatory G protein (Gs) in 231-ZIP9 cells which is consistent with the finding that treatment with the adenylyl cyclase (AC) stimulator forskolin, further increased migration marker expression and migration compared to vehicle controls, whereas the PKA inhibitor, H-89, attenuated these responses as well as the increase in free zinc concentrations. Knockdown of ZIP9 expression in MDA-MB-468 cells which express relatively high levels of ZIP9 with siRNA decreased cell migration and invasion as well as free zinc levels, confirming a role of unliganded ZIP9 in migration in another breast cancer cell line. Treatment of 231-ZIP9 cells with 100 nM testosterone further stimulated CYR61 and MYL9 expression and cell migration which were blocked by co-treatment with a Gs inhibitor and TPEN. The results indicate that high ZIP9 expression in breast cancer cells causes androgen-independent increased cell migration and invasion which in 231-ZIP9 cells is through Gs/mAC/PKA-mediated increases in intracellular zinc concentrations and upregulation of MYL9 and CYR-61 expression. Furthermore, the results suggest that testosterone-dependent signaling through ZIP9 acts through the same pathways to further enhance signaling and migration. Presentation: Saturday, June 17, 2023

Zinc transporter ZIP9 is also a membrane androgen receptor that mediates androgen-dependent zinc and G-protein signaling to modulate tumorigenic responses in cancer cells. It is unclear whether unliganded ZIP9 causes similar responses. ZIP9 overexpression in MDA-MB-231 breast cancer cells (ZIP9 cells) increased zinc levels and cell migration/invasion which was mimicked with a zinc ionophore and attenuated with a zinc chelator, suggesting these tumorigenic responses are zinc-dependent. Expression of migration markers MYL9 and CYR61 was elevated in ZIP9 cells and further increased together with cell migration by forskolin treatment and blocked with H-89, indicating they are mediated through an AC/PKA pathway. Knockdown of ZIP9 expression in MDA-MB-468 cells decreased cell migration/invasion, migration markers and zinc levels, confirming similar roles of unliganded ZIP9 in another breast cancer cell line. Testosterone treatment further increased migration, biomarker expression and zinc in ZIP9 cells, suggesting it may act through similar pathways to induce tumorigenic responses.

ZIP9 was recently characterized as a membrane androgen receptor in Atlantic croaker granulosa/theca (G/T) cells where it mediates androgen-induced apoptosis in vitro, but the physiological significance of this action has remained unclear. In the current study, we utilized ZIP9 knockout (zip9-/-) zebrafish to investigate the role of ZIP9-mediated androgen-induced G/T cell apoptosis in vivo. We first confirmed ZIP9 mediates apoptosis of zebrafish G/T cells in vitro. Testosterone increased apoptosis, intracellular free zinc, and expression of pro-apoptotic members bax and p53 in wildtype and zip9+/+ zebrafish G/T cells, but not in ZIP9 knockout and knockdown cell models. We hypothesized ZIP9-mediated G/T cell apoptosis may be involved in post-ovulatory follicle (POF) breakdown in vivo. Post ovulation, zip9, bax, and p53 were upregulated in zip9+/+ but not in zip9-/- ovaries. Immunoreactivity of cleaved caspase 3 was also higher in POFs from zip9+/+ ovaries compared to zip9-/-, and POF breakdown was significantly delayed in zip9-/- fish compared to zip9+/+ counterparts. To determine the detrimental consequences of delayed POF breakdown in the zip9-/- model, fish were challenged with repeated ovulation induction. After the challenge, zip9-/- fish exhibited abnormal ovarian lesions that contained debris consistent with atretic or necrotic cellular material. However, no abnormalities were observed in zip9+/+ fish ovaries, indicating that the abnormal phenotype is due to the loss of ZIP9. This study demonstrates an important role for ZIP9 in mediating POF breakdown and maintaining tissue remodeling and homeostasis in the teleost ovary and indicates a role for the ZIP9-mediated androgen-induced apoptotic response in vivo.

The 7-transmembrane architecture of adiponectin receptors (AdipoRs), determined from their X-ray crystal structures, was used for homology modeling of another progesterone and adipoQ receptor (PAQR) family member, membrane progesterone receptor alpha (mPRα). The mPRα model identified excess positively charged residues on the cytosolic side, suggesting it has the same membrane orientation as AdipoRs with an intracellular N-terminus. The homology model showed identical amino acid residues to those forming the zinc binding pocket in AdipoRs, which strongly implies that zinc is also present in mPRα. The homology model showed a critical H-bond interaction between the glutamine (Q) residue at 206 in the binding pocket and the 20-carbonyl of progesterone. Mutational analysis showed no progesterone binding to the arginine (R) 206 mutant and modeling predicted this was due to the strong positive charge of arginine stabilizing the presence of an oleic acid (C18:1) molecule in the binding pocket, as observed in the X-rays of AdipoRs. High Zn2+ concentrations are predicted to form a salt with the carboxylate group of the oleic acid, thereby eliminating its binding to the free fatty acid (FFA) binding pocket, and allowing progesterone to bind. This is supported by experiments showing 100 µM Zn2+ addition restored [3H]-progesterone binding of the Q206R mutant to levels in WT mPRα and increased [3H]-progesterone binding to mPRγ and AdipoR1 which have arginine residues in this region. The model predicts hydrophobic interactions of progesterone with amino acid residues surrounding the binding pocket, including valine 146 in TM3, which when mutated into a polar serine resulted in a complete loss of [3H]-progesterone binding. The mPRα model showed there is no hydrogen bond donor in the vicinity of the 3-keto group of progesterone and ligand structure-activity studies with 3-deoxy steroids revealed that, unlike the nuclear progesterone receptor, the 3-carbonyl oxygen is not essential for binding to mPRα. Interestingly, the small synthetic AdipoR agonist, AdipoRon, displayed binding affinity for mPRα and mimicked progesterone signaling, whereas D-e-MAPP, a ceramidase inhibitor, blocked progesterone signaling. Thus, critical residues around the binding pocket and steroid structures that bind mPRα, as well as similarities with AdipoRs, can be predicted from the homology model.

Cholesterol affects the proliferation of breast cancer (BC) and in particular of estrogen receptor-negative (ER−) BC. Cholesterol is converted to 27-hydroxycholesterol (27HC), which promotes the growth of ER+ BC. Potentially, 27HC can be involved in cholesterol-dependent ER− BC proliferation. Stable MDA-MB-231 silenced clones for CYP7B1 (27HC metabolizing enzyme) show an increased basal proliferation rate, which is not observed in the presence of lipoprotein-deprived serum. Furthermore, the treatment of SKBR3, MDA-MB-231 and MDA-MB-468 with 27HC increased cell proliferation that was prevented by G15, a selective G Protein-Coupled Estrogen Receptor (GPER) inhibitor, suggested this receptor to be a potential 27HC target. Binding experiments demonstrate that 27HC is a new ligand for GPER. We show that ERK1/2 and NFκB are part of the 27HC/GPER pathway. The stable silencing of GPER prevents NFκB activation and reduces basal and 27HC-dependent tumor growth. Additionally, conditioned medium from ER− BC cells treated with 27HC promotes tube formation, which does not occur with CM from GPER silenced cells. Collectively, these data demonstrate that cholesterol conversion into 27HC promotes ER− BC growth and progression, and the expression of GPER is required for its effects.

Progesterone causes vascular smooth muscle cell relaxation through membrane progesterone receptors (mPRs), which are members of the progestin and adipoQ receptor (PAQR) family, and nuclear PRs (nPRs). However, beneficial vascular effects of progesterone in preventing pre-atherosclerosis and the involvement of mPRs and nPRs remain unclear. The results show short- to long-term treatments with 100 nM progesterone (P4) and specific agonists for mPRs, OD 02-0, and nPRs, R5020, inhibited pre-atherosclerotic events in human umbilical vein endothelial cells (HUVECs), decreasing focal adhesion (FA) by monocytes, FA signaling, HUVEC migration and invasion, and vinculin expression. Progesterone and OD 02-0, but not R5020, inhibited phosphorylation of Src and focal adhesion kinase, critical kinases of FA signaling, within 20 min and migration and invasion of HUVECs and monocyte adhesion after 3 h. These inhibitory P4 and 02-0 effects were attenuated with MAP kinase and Pi3k inhibitors, indicating involvement of these kinases in this mPR-mediated action. However, after 16 h, OD 02-0 was no longer effective in inhibiting FA signaling, while both progesterone and R5020 decreased the activity of the two kinases. Knockdown of receptor expression with siRNA confirmed that mPRα mediates short-term and nPR long-term inhibitory effects of progesterone on FA signaling. Thus, progesterone inhibition of FA signaling and pre-atherosclerosis is coordinated through mPRα and nPRs.

Schwann cells and neuroregeneration: Peripheral nerve injury is a common cause of morbidity, which affects millions of people worldwide. The peripheral nervous system, differently from the central nervous system, has an intrinsic ability to regenerate after injury. However, in most cases the regenerative outcome is not completely satisfactory, in particular for long-gap peripheral nerve injuries in which the microsurgical approach is not possible. In these cases, the current research effort is mostly aimed at the identification of pharmacological and/or cell therapy approaches that, coupled with the use of biomaterial conduits, provide scaffold, mechanical support and guidance to the regeneration process, and can increase regeneration speed and efficiency (Faroni et al., 2015).

(-)-Epicatechin, a flavonoid present in high concentrations in foods such as green tea and cocoa, exerts beneficial and protective effects in numerous disease models, including anti-tumorigenesis and apoptosis in human breast and prostate cancer cells. Potential interactions of (-)-epicatechin and (+)-catechin with the membrane androgen receptor, ZIP9 (SLC39A9), which mediates androgen induction of apoptosis in these cancer cells, were investigated. Both (-)-epicatechin and (+)-catechin were effective competitors of [3H]-testosterone binding to PC-3 prostate cancer cells (nuclear androgen receptor-negative, nAR-null) overexpressing ZIP9 (PC3-ZIP9), with relative binding affinities of 75 % and 28 % that of testosterone, respectively. (-)-Epicatechin (200 nM) mimicked the effects of 100 nM testosterone in inducing apoptosis of PC3-ZIP9 cells, whereas (+)-catechin (concentration range 200 nM-1000 nM) did not significantly increase apoptosis and instead blocked the apoptotic response to testosterone. (-)-Epicatechin also activated androgen-dependent ZIP9 signaling pathways, inducing decreases in cAMP production and elevating intracellular free zinc levels, while (+)-catechin typically lacked these actions. Both (-)-epicatechin and (+)-catechin also bound to cell membranes of MDA-MB-468 breast cancer cells (nAR-null, high ZIP9 expression). MDA-MB-468 cells showed similar apoptotic, cAMP, and free zinc signaling responses to (-)-epicatechin to those observed in PC3-ZIP9 cells, as well as antagonism by (+)-catechin of testosterone-induced apoptosis and modulation of cAMP and caspase-3 levels. Moreover, knockdown of ZIP9 expression in MDA-MB-468 cells with siRNA decreased specific [3H]-testosterone binding of both catechins and blocked the apoptotic and free zinc responses to testosterone and (-)-epicatechin. The results indicate (-)-epicatechin is a potent ZIP9 agonist in breast and prostate cancer cells.

The role G-protein coupled estrogen receptor (GPER) plays in vertebrate reproduction remains controversial. To investigate GPER’s reproductive role, we generated a gper zebrafish mutant line (gper−/−) using TALENs. Gper mutant females exhibited reduced fertility with a 40.85% decrease in embryo production which was associated with a significant decrease in the number of Stage V (730–750 μm) ovulated oocytes. Correspondingly, the number of early vitellogenic follicles (Stage III, 400–450 µm) in gper−/− ovaries was greater than that in wildtypes (wt), suggesting that subsequent follicle development was retarded in the gper−/− fish. Moreover, plasma vitellogenin levels were decreased in gper−/− females, and epidermal growth factor receptor (Egfr) expression was lower in Stage III vitellogenic oocytes than in wt counterparts. However, hepatic nuclear estrogen receptor levels were not altered, and estrogen levels were elevated in ovarian follicles. These results suggest that Gper is involved in the control of ovarian follicle development via regulation of vitellogenesis and Egfr expression in zebrafish.

The rapid effects of progesterone on cytosolic Ca 2+ levels and relaxation of VSMCs through mPRα involve regulation of the functions of SERCA2 and PLB through Gi, MAP kinase, and Akt signaling pathways and downregulation of RhoA activity.

We have previously shown that nitric oxide synthase (NOS, an enzyme) is significantly increased during hypoxic stress in Atlantic croaker brains and modulated by an antioxidant (AOX). However, the influence of NOS and AOX on cytochrome P450 aromatase (AROM, CYP19a1, an enzyme) activity on vertebrate brains during hypoxic stress is largely unknown. In this study, we characterized brain AROM (bAROM, CYP19a1b) cDNA in croaker and examined the interactive effects of hypoxia and a NOS-inhibitor or AOX on AROM activity. The amino acid sequence of croaker bAROM cDNA is highly homologous (76–80%) to other marine teleost bAROM cDNAs. Both real-time PCR and Northern blot analyses showed that bAROM transcript (size: ∼2.8 kb) is highly expressed in the preoptic-anterior hypothalamus (POAH). Hypoxia exposure (dissolved oxygen, DO: 1.7 mg/L for 4 weeks) caused significant decreases in hypothalamic AROM activity, bAROM mRNA and protein expressions. Hypothalamic AROM activity and mRNA levels were also decreased by pharmacological treatment with N -ethylmaleimide (NEM, an alkylating drug that modifies sulfhydryl groups) of fish exposed to normoxic (DO: ∼6.5 mg/L) conditions. On the other hand, treatments with N ω-nitro- L -arginine methyl ester (NAME, a competitive NOS-inhibitor) or vitamin-E (Vit-E, a powerful AOX) prevented the downregulation of hypothalamic AROM activity and mRNA levels in hypoxic fish. Moreover, NAME and Vit-E treatments also restored gonadal growth in hypoxic fish. Double-labeled immunohistochemistry results showed that AROM and NOS proteins are co-expressed with NADPH oxidase (generates superoxide anion) in the POAH. Collectively, these results suggest that the hypoxia-induced downregulation of AROM activity in teleost brains is influenced by neuronal NOS activity and AOX status. The present study provides, to the best of our knowledge, the first evidence of restoration of AROM levels in vertebrate brains by a competitive NOS-inhibitor and potent AOX during hypoxic stress.

The neurosteroids progesterone and allopregnanolone regulate numerous neuroprotective functions in neural tissues including inhibition of epileptic seizures and cell death. Many of progesterone's actions are mediated through the nuclear progesterone receptor (PR), while allopregnanolone is widely considered to be devoid of hormonal activity and instead acts through modulation of GABA-A receptor activity. However, allopregnanolone can also exert hormonal actions in neuronal cells through binding and activating membrane progesterone receptors (mPRs) belonging to the progestin and adipoQ receptor (PAQR) family. The distribution and functions of the five mPR subtypes (α, β, γ, δ, ε) in neural tissues are briefly reviewed. mPRδ has the highest binding affinity for allopregnanolone and is highly expressed throughout the human brain. Low concentrations (20 nM) of allopregnanolone act through mPRδ to stimulate G protein (Gs)-dependent signaling pathways resulting in reduced cell death and apoptosis in mPRδ-transfected cells. The 3-methylated synthetic analog of allopregnanolone, ganaxolone, is currently undergoing clinical trials as a promising GABA-A receptor-selective antiepileptic drug (AED). New data show that low concentrations (20 nM) of ganaxolone also activate mPRδ signaling and exert anti-apoptotic actions through this receptor. Preliminary evidence suggests that ganaxolone can also exert neuroprotective effects by activating inhibitory G protein (Gi)-dependent signaling through mPRα and/or mPRβ in neuronal cells. The results indicate that mPRs are likely intermediaries in multiple actions of natural and synthetic neurosteroids in the brain. Potential off-target effects of ganaxolone through activation of mPRs in patients receiving long-term treatment for epilepsy and other disorders should be considered and warrant further investigation.

Yellowfin porgy a protandrous teleost, exhibits asynchronous oocyte development and multiple spawning. Seasonal profiles of plasma estradiol-17β (E2) levels showed a peak in three-year-old females during the spawning season, when batches of fully-grown oocytes undergo final oocyte maturation (FOM). Because E2 has been shown to inhibit FOM via the G protein-coupled estrogen receptor (Gper) in several teleost species, we investigated the role of this "paradoxical" increase in E2 during FOM in yellowfin porgy. In vivo treatment with a GnRH-agonist stimulated germinal vesicle breakdown (GVBD) and increased E2 plasma levels, and ovarian cyp19a1a transcripts, confirming the increase in E2 production at the time of FOM. Ovarian transcripts of gper peaked at the time of FOM, indicating an increase in ovarian responsiveness to Gper-mediated E2 effects. In vitro, E2 and the Gper agonist, G-1, inhibited the stimulatory effect of maturation-inducing steroids (MIS) on GVBD, while an aromatase inhibitor enhanced the MIS effect, in agreement with a physiological inhibitory role of E2 on FOM via Gper. Immunohistological studies showed that the Gper protein was specifically located on the oocyte plasma membrane. Ovarian membranes displayed high-affinity and limited-capacity specific [3H]-E2 receptor binding which was displaced by G-1, characteristic of Gper. Expression of gper increased at the time of FOM in mid-vitellogenic oocytes, but not in larger oocytes undergoing GVBD. These results suggest increases in both E2 production and E2 responsiveness via Gper upregulation in mid-vitellogenic oocytes, may maintain meiotic arrest in this oocyte stage class during the period when full-grown oocytes are undergoing FOM. This study indicates a critical involvement of E2 in the control of asynchronous oocyte maturation and the multiple spawning pattern in Sparidae.