cortodoxone and 17-20-21-trihydroxy-4-pregnen-3-one

Plasma levels of reproductive and thyroid hormones were measured in captive striped bass females during postvitellogenesis and the spawning period (March-June). Circulating gonadotropin II (GtH II), 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P), and 17,20beta, 21-trihydroxy-4-pregnen-3-one (17,20beta,21-P) remained low and unchanged in nonmaturing females, while 17beta-estradiol (E2) and testosterone (T) declined throughout postvitellogenesis. Plasma thyroxine (T4) declined significantly in mid-April, while triiodothyronine (T3) increased in mid-May. The only female that ovulated spontaneously had markedly different E2, T, and T3 profiles during postvitellogenesis, and had a surge in plasma GtH II during final oocyte maturation (FOM). The lack of a GtH II surge is presumably responsible for the absence of FOM, but earlier, and as of yet unknown, endocrine disruptions during postvitellogenesis may determine the female's ability to undergo FOM. Treatment of females with a gonadotropin-releasing hormone agonist (GnRHa)-delivery system induced FOM and ovulation within 3 and 10 days, respectively, and resulted in the production of fertile eggs. Plasma GtH II increased continually after GnRHa implantation, even in the presence of declining GnRHa plasma levels. Plasma E2 increased first and peaked prior to FOM, whereas T peaked at the peripheral germinal vesicle (GV) stage. Plasma 17,20beta-P and 17,20beta,21-P increased dramatically at the GV breakdown (GVBD) stage. Plasma T4 was unaffected by the GnRHa treatment, whereas T3 decreased after GnRHa implantation and remained low throughout FOM. Based on the observed hormonal profiles, FOM can be separated into an early phase (lipid-droplet coalescence, GV migration) associated with E2 and T elevations, and a late phase (yolk-globule coalescence, GVBD) associated with 17,20beta-P and 17,20beta,21-P elevation.

Topics: Animals; Animals, Wild; Bass; Cortodoxone; Estradiol; Female; Gonadal Steroid Hormones; Gonadotropin-Releasing Hormone; Gonadotropins, Pituitary; Hydroxyprogesterones; Oogenesis; Testosterone; Thyroid Hormones; Thyroxine; Triiodothyronine; Vitellogenesis

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 17,20β,21-trihydroxy-4-pregnen-3-one (20β-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 20β-S treatment. These results demonstrate that progestin-mediated hypermotility via mPRα in croaker sperm involves activation of the Egfr, ErbB2 and Mapk pathways.

Topics: Animals; Cortodoxone; ErbB Receptors; Fish Proteins; Hydroxamic Acids; Indoles; Male; Mitogen-Activated Protein Kinases; Perciformes; Progestins; Pyrimidines; Receptor, ErbB-2; Signal Transduction; Sperm Motility

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, 17,20β, 21-trihydroxy-4-pregnen-3-one (20β-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 20β-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.

Topics: Animals; Cell Membrane; Cortodoxone; Cyclic AMP; Fertilization; Flounder; Gonadotropin-Releasing Hormone; GTP-Binding Protein alpha Subunits, Gs; Humans; Injections; Male; Norprogesterones; Progestins; Receptors, Progesterone; Sperm Motility; Spermatozoa; Up-Regulation

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 17,20β,21-trihydroxy-4-pregnen-3-one (20β-S) during thin-layer chromatography. Treatment of flounder sperm with 5 nM-100 nM 20β-S significantly increased sperm velocity in vitro, whereas 17,20β-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 20β-S was identified on sperm membranes. Treatment of sperm membranes with guanosine 5'-(3-O-thio)triphosphate reduced [(3)H]-20β-S binding, suggesting the 20β-S receptor couples to a G protein. The membrane adenylyl cyclase inhibitor 2',5'-dideoxyadenosine blocked 20β-S-induced sperm hypermotility, indicating 20β-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 20β-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.

Topics: Amino Acid Sequence; Animals; Cortodoxone; Dideoxyadenosine; Flounder; Male; Molecular Sequence Data; Receptors, Progesterone; Sequence Alignment; Sperm Motility

Although there is substantial evidence that membrane progestin receptors (mPRs) perform a critical physiological role in meiotic maturation of fish oocytes, it is unknown whether they are also intermediaries in progestin signaling in the surrounding follicular cells. Here, we show that mPRα protein is located on the plasma membranes of both granulosa and theca cells (G/T cells) isolated from Atlantic croaker ovaries and is associated with the presence of a single high affinity, limited capacity, pertussis toxin-sensitive, specific progestin [17,20β,21-trihydroxy-4-pregnen-3-one (20β-S)] membrane binding site with the characteristics of mPRα. Treatment of G/T cells with 20β-S caused rapid G protein activation and a transient, pertussis toxin-sensitive, decrease in cAMP levels, whereas the selective nuclear progesterone receptor agonist, R5020, did not cause G protein activation, consistent with previous reports on mPRα signaling. 20β-S treatment decreased serum starvation-induced cell death in both G/T cells and in seatrout mPRα-transfected MDA-MB-231 cells, whereas R5020 was ineffective. Moreover, a selective mPRα agonist, 10-ethenyl-19-norprogesterone, mimicked the protective action of 20β-S against cell death, which was lost upon knockdown of mPRα protein but not after progesterone receptor knockdown, further demonstrating an involvement of mPRα. Signaling molecules involved in inhibition of apoptosis, Erk and serine-threonine kinase, were activated in G/T cells by 20β-S, which suggests a potential mechanism for mPRα inhibition of apoptosis. This is the first study to demonstrate endogenous mPR signaling in the ovarian follicle and to suggest a novel physiological role for mPRα in mediating the antiapoptotic actions of progestins in ovarian follicle cells.

Topics: Animals; Apoptosis; Cell Death; Cells, Cultured; Coculture Techniques; Cortodoxone; Extracellular Signal-Regulated MAP Kinases; Female; Granulosa Cells; Perciformes; Progestins; Proto-Oncogene Proteins c-akt; Receptors, Progesterone; RNA, Small Interfering; Theca Cells

Recent studies have shown that chronic hypoxia exposure impairs reproduction in fish by interfering with endocrine function, although the mechanisms of endocrine disruption remain unclear. The effects of chronic exposure (4 or 10 weeks) to hypoxia (dissolved oxygen, DO: 1.7 mg L(-1)) on gamete maturation and its endocrine control, as well as the consequences for reproductive success, were investigated in Atlantic croaker (Micropogonias undulatus). Circulating levels of the progestin hormone that induces gamete maturation, 17,20beta,21-trihydroxy-4-pregnen-3-one (20beta-S), were significantly decreased in croaker of both sexes chronically exposed to hypoxia and were associated with impairment of oocyte meiotic maturation and sperm motility. Interestingly, expression of the novel membrane receptor mediating these nongenomic 20beta-S actions, membrane progestin receptor alpha (mPRalpha), was significantly decreased on oocyte and sperm plasma membranes of hypoxia-exposed fish. Hypoxia-induced impairment of gamete maturation was accompanied with a dramatic decline in the percent fertilized eggs in a spawning trial. Moreover, the fertilized eggs from hypoxia-exposed donors displayed decreased hatching success and larval survival. The results suggestthat nongenomic progestin signaling controlling the final stages of the reproductive cycle in fish is impaired under hypoxic conditions.

Topics: Animals; Cortodoxone; Endocrine Disruptors; Female; Hypoxia; Male; Oocytes; Perciformes; Progestins; Reproduction; Sperm Maturation; Spermatozoa; Water Pollutants, Chemical

Recent studies have shown that several environmental chemicals which disrupt classical genomic steroid actions can also interfere with nongenomic steroid actions initiated at the cell surface. The effects of bisphenol-A and atrazine on the nongenomic actions of a progestin, 17,20 beta,21-trihydroxy-4-pregnen-3-one (20 beta-S), on oocyte maturation (OM) were investigated an in vitro Atlantic croaker (Micropogonias undulatus) oocyte bioassay. Treatment of croaker follicle-enclosed oocytes with either bisphenol-A or atrazine blocked OM in response to 20 beta-S in a concentration-dependent manner at 10-25 microM (2.2-5.7 ppm) and higher concentrations. These compounds were also effective competitors at concentrations of 10(-6)-10(-5)M of [3H]-20 beta-S binding to the ovarian progestin membrane receptor that mediates the OM response to 20 beta-S. The results suggest that diverse classes of environmental chemicals can disrupt nongenomic progestin actions via receptor-mediated mechanisms.

Topics: Animals; Atrazine; Benzhydryl Compounds; Cortodoxone; Female; Oocytes; Ovary; Perciformes; Phenols; Progestins; Protein Binding; Receptors, Progesterone; Water Pollutants, Chemical

The identity of the maturation-inducing steroid (MIS) in black porgy, Acanthopagrus schlegeli, a marine protandrous teleost, is unknown. Previous studies demonstrated that two teleost MISs, the progestins 17,20beta,21-trihydroxy-4-pregnen-3-one (20beta-S) and 17,20beta-dihydroxy-4-pregnen-3-one (DHP) can induce maturation of black porgy oocytes in vitro. The purpose of the present study was to identify the major progestin produced during oocyte maturation (OM) in black porgy and investigate whether its secretion increases during this process. Females were injected twice with a LHRH analog to induce OM. Ovarian follicles undergoing OM were incubated in vitro with tritiated [3H]pregnenolone precursor and the tritiated products were extracted, purified, and identified by HPLC, TLC, acetylation, and recrystallization. Significant amounts of tritiated products were biosynthesized from [3H]pregnenolone that co-migrated with 20beta-S but not with DHP on HPLC and TLC. Similar TLC profiles were obtained with the tritiated products isolated from the HPLC/TLC 20beta-S fraction and standard 20beta-S after the acetylation reaction. The identity of the tritiated products as 20beta-S was confirmed by recrystallization. 20beta-S had a slightly higher potency than DHP in the inducing in vitro final oocyte maturation. Plasma 20beta-S concentrations increased significantly during the oocyte maturation after injection with a LHRH analog. The present data suggest that 20beta-S is the MIS in black porgy.

Topics: Acetylation; Animals; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Cortodoxone; Crystallization; Female; Gonadotropin-Releasing Hormone; Maturation-Promoting Factor; Oocytes; Perciformes

A combination of recombinant human (rh) insulin-like growth factor-I (IGF-I) (25 nM) and the maturation-inducing hormone (MIH), 17,20beta,21-trihydroxy-4-pregnen-3-one (20beta-S; 72.5 nM), induced germinal vesicle breakdown (GVBD) in ovarian follicles of white bass incubated in vitro, whereas a four times greater concentration of each hormone was ineffective alone. These results indicate that IGF-I induces oocyte maturational competence (OMC) but not meiotic resumption in white bass. Culture medium concentrations of 20beta-S remained below detection limits for ovarian fragments incubated with rhIGF-I. Actinomycin D blocked GVBD in response to hCG but not to rhIGF-I plus 20beta-S, suggesting that IGF-I requires de novo translation but not transcription to induce OMC. Gap junction uncouplers, 1-octanol and 1-heptanol, and the phosphatidylinositiol 3-kinase (PI 3-K) inhibitors, wortmannin and LY 294002, attenuated hCG-, 20beta-S-, and rhIGF-I plus 20beta-S-induced GVBD. Although these inhibitors reduced hCG-induced progestin release, PI 3-K inhibitors did not alter MIH synthesis in some incubations and addition of 20beta-S to the incubations did not fully overcome the effects of either class of inhibitors, suggesting that decreasing MIH production is not their only inhibitory effect on gonadotropin (GtH) action. Our data suggest that gap junctions and PI 3-K activity are necessary for GtH and IGF-I to induce and maintain OMC in white bass. The induction of OMC but not meiotic resumption by IGF-I in white bass, compared with the induction of meiotic resumption but not OMC by IGF-I discovered in the congeneric striped bass suggests rapid evolution of the reproductive actions of IGF-I among temperate basses (genus Morone).

Topics: 1-Octanol; Androstadienes; Animals; Bass; Chromones; Cortodoxone; Cycloheximide; Dactinomycin; Enzyme Inhibitors; Female; Gap Junctions; Heptanol; Humans; In Vitro Techniques; Insulin; Insulin-Like Growth Factor I; Meiosis; Morpholines; Oocytes; Ovarian Follicle; Phosphoinositide-3 Kinase Inhibitors; Recombinant Proteins; Wortmannin

In vitro studies of ovarian follicle maturation and ovulation in teleost fishes typically are conducted within a narrow range (7.5-7.8) of constant external (medium) pH, although there is evidence that pH can influence ovulation. Therefore, this study with Atlantic croaker investigated the effects of external pH on hormonally regulated in vitro maturation and ovulation as well as changes in the pH of ovarian fluid during in vivo maturation and ovulation. For the in vitro experiments, follicles were first incubated with human chorionic gonadotropin (hCG) to induce maturational and ovulatory competencies, and then with maturation-inducing hormone (MIH) to induce completion of maturation and ovulation. At a constant external pH within the range of 7.0-8.2, the lower pH levels (7.0-7.3) generally inhibited or slowed down hormonally induced maturation and ovulation whereas higher pH (7.6-8.2) facilitated these processes. When ovarian follicles were incubated at a constant pH of 7.6 during the priming incubation with hCG, changing the external pH during the incubation with MIH had relatively little effect on oocyte maturation or ovulation. Thus, the inhibitory effect of constant low levels of external pH (7.0-7.3) on maturation and ovulation may be primarily due to disruptions in the gonadotropin-dependent acquisition of maturational and ovulatory competencies. The pH of ovarian fluid remained constant at 8.5 during in vivo ovarian follicle maturation and ovulation. Subsequent in vitro tests showed that external pH of 8.5 enhances hormonally induced maturation and ovulation relative to pH of 7.6. These observations suggest that attention should be paid to the pH of incubation media used in basic research and in biotechnological applications relying on in vitro maturation and ovulation in teleosts. Further, an understanding of the physiological significance of the enhancing effect of alkaline pH on maturation and ovulation will require determination of the intrafollicular pH around the oocyte during the periovulatory period.

Topics: Animals; Chorionic Gonadotropin; Cortodoxone; Dose-Response Relationship, Drug; Female; Follicular Phase; Hydrogen-Ion Concentration; Oocytes; Ovarian Follicle; Ovulation; Perciformes

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.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Animals; Cortodoxone; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic Nucleotide Phosphodiesterases, Type 3; Cyclic Nucleotide Phosphodiesterases, Type 4; Enzyme Activation; Enzyme Inhibitors; Female; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Oncogene Protein v-akt; Oocytes; Perciformes; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Signal Transduction; Steroids

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.

Topics: Adenylyl Cyclases; Animals; Cortodoxone; Enzyme Activation; Female; GTP-Binding Protein alpha Subunits, Gi-Go; Oocytes; Oogenesis; Perciformes; Pertussis Toxin

TGF-beta is a multifunctional growth factor involved in regulating a variety of cellular activities. Unlike mammals, the function of TGF-beta in the reproduction of lower vertebrates, such as fish, is not clear. Recently, we showed that TGF-beta1 inhibits gonadotropin- and 17alpha, 20beta-dihydroxyprogesterone (DHP)-induced maturation in zebrafish. The aim of the present study was to investigate the mechanisms underlying this action.. To determine if the effect of TGF-beta1 on oocyte maturation involves transcription and/or translation, ovarian follicles were pre-treated with actinomycin D, a blocker of transcription, and cyclohexamide, an inhibitor of translation, and incubated with hCG or DHP, either alone or in combination with TGF-beta1 and oocyte maturation scored. To determine the effect of TGF-beta1 on mRNA levels of several key effectors of oocyte maturation, three sets of experiments were performed. First, follicles were treated with control medium or TGF-beta1 for 2, 6, 12, and 24 h. Second, follicles were treated with different concentrations of TGF-beta1 (0 to 10 ng/ml) for 18 h. Third, follicles were incubated with hCG in the absence or presence of TGF-beta1 for 18 h. At the end of each experiment, total RNA was extracted and reverse transcribed. PCR using primers specific for 20beta-hydroxysteroid dehydrogenase (20beta-HSD) which is involved in DHP production, follicle stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR), the two forms of membrane progestin receptor: mPR-alpha and mPR-beta, as well as GAPDH (control), were performed.. Treatment with actinomycin D, a blocker of transcription, reduced the inhibitory effect of TGF-beta1 on DHP-induced oocyte maturation, indicating that the inhibitory action of TGF-beta1 is in part due to regulation of gene transcription. Treatment with TGF-beta1 caused a dose and time-dependent decrease in mRNA levels of 20beta-HSD, LHR and mPR-beta in follicles. On the other hand, TGF-beta1 had no effect on mPR-alpha mRNA expression and increased FSHR mRNA levels. Furthermore, hCG upregulated 20beta-HSD, LHR and mPR-beta mRNA levels, but this stimulatory effect was blocked by TGF-beta1.. These findings suggest that TGF-beta1 acts at multiple sites, including LHR, 20beta-HSD and mPR-beta, to inhibit zebrafish oocyte maturation.

Topics: Animals; Chorionic Gonadotropin; Cortisone Reductase; Cortodoxone; Cycloheximide; Dactinomycin; Down-Regulation; Female; Gene Expression; Hydroxyprogesterones; Oocytes; Ovarian Follicle; Receptors, FSH; Receptors, LH; Receptors, Progesterone; Transforming Growth Factor beta; Transforming Growth Factor beta1; Zebrafish; Zebrafish Proteins

Final oocyte maturation (FOM) is a process involving a complex set of genetical, biochemical, and morphological mechanisms. FOM involves the shift of a post-vitellogenic follicle to a pre-ovulated oocyte, which is necessary for fertilization by spermatozoan to occur. This process is regulated by a maturation-inducing steroid (MIS) at the follicular level. In other species of scienids fish the MIS, a hydroxilated derivatives of progestagen 17, 20beta, 21-trihydroxy-4-pregnen-3-one (20beta-S), was identified. Although Micropogonias furnieri is the second fishery resource of Uruguay, basic knowledge about its endocrine process is very scarce. The aim of this work was to investigate what steroids are synthesized in vitro by the oocyte follicle of M. furnieri during the maturation process. Fragments of ovary (1 g) in three stages: post-vitellogenic (PV), maturing (Mtg), and mature (M) were incubated with 1 microg x g(-1) of tritiated progesterone (P) at 30, 60, and 180 min. After extraction with ethanol and dichloromethane, steroid metabolites were purified by TLC and rpHPLC. Two progesterone derivatives with identical chromatographic properties of 20beta-S and 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P) were purified. In other Teleost fish these steroids are biologically active as MIS. The 17,20beta-P was clearly detected in Mtg and M stages and confirmed by enzymatic oxidation with enzyme 20beta-HSD. The 20beta-S was strongly detected in all Mtg oocytes. The results do not corroborate 20beta-S as a major hormone synthesized in the ovary in FOM as occurs in other scienid fish. A differential steroid synthesis in the advanced oocyte stages suggests that the 20beta-S is acting as a MIS in M. furnieri.

Topics: Animals; Chromatography, Thin Layer; Cortodoxone; Female; Fishes; Oocytes; Steroids; Time Factors

Behavioral, biochemical, and electrophysiological studies suggest that the trihydroxylated progestin steroid, 4-pregnen-17,20beta,21-triol-3-one (20beta-S) stimulates oocyte maturation and pheromone release in the Eurasian ruffe, a freshwater percid fish. Behavioral observations found that female ruffe undergoing oocyte maturation (OM) release a pheromonal cue that stimulates swimming activity and social interactions among conspecific males. Neither vitellogenic nor ovulated females released the cue. Pheromone production was directly associated with elevated plasma levels of 20beta-S in maturing female ruffe which in vitro incubation suggested to be a possible maturation-inducing hormone (MIH) in this species along with 4-pregnen-17,20beta-diol-3-one (17,20betaP). However, neither of these steroids appear to be the pheromone because electrophysiological and behavioral studies found them to lack olfactory (EOG) and behavioral activity. Instead, studies of the odor of steroid-injected fish suggest the pheromone is a metabolite of 20beta-S. In particular, inter-peritoneal injection of 20beta-S (but not 17,20betaP) consistently induced release of a urinary cue with strong behavioral activity. The pheromone may be a highly polar and novel metabolite because it could not be extracted using octadecylsilane resin (C18) which has proven effective for other teleost hormonal pheromones.

Topics: Animals; Behavior, Animal; Cortodoxone; Cues; Female; Hormones; Injections, Intraperitoneal; Male; Oogenesis; Perches; Sex Attractants; Smell; Urine

The present study examined diurnal cycles of oocyte development and maturation in the kyusen wrasse, Halichoeres poecilopterus, and investigated the sensitivity of oocytes to maturation-inducing hormone (MIH) and gonadotropic hormone (GTH). Female fish were sampled at fixed intervals throughout the day, revealing that final oocyte maturation and ovulation were completed by 6:00 hr, and that spawning occurred daily between 6:00 and 9:00 hr. In vitro experiments showed that the steroids 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P) and 17,20beta,21-trihydroxy-4-pregnen-3-one (20beta-S) were equally potent and highly effective inducers of germinal vesicle breakdown (GVBD) in kyusen wrasse oocytes. Additionally, circulating levels of 17,20beta-P and 20beta-S increased around the time of GVBD and ovulation, suggesting that 17,20beta-P and 20beta-S act as MIHs in the kyusen wrasse. Moreover, in vitro experiments clearly showed that kyusen wrasse oocytes had a daily developmental cycle of GTH and MIH sensitivity, and that oocytes that completed vitellogenesis acquired GTH-induced maturational competence. An endogenous GTH surge likely occurs between 12:00 and 15:00 hr, and this daily pre-maturational GTH surge probably controls the diurnal maturation cycles of kyusen wrasse oocytes.

Topics: Animals; Circadian Rhythm; Cortodoxone; Female; Fishes; Gonadotropins; Hormones; Hydroxyprogesterones; Maturation-Promoting Factor; Oocytes; Ovulation

The effects of treatment with the maturation-inducing steroid 17,20beta,21-trihydroxy-4-pregnen-3-one (20beta-S) on luteinizing hormone-releasing hormone analog (LHRHa)-induced LH secretion were examined during several phases of the gonadal cycle in Atlantic croaker, Micropogonias undulatus. 20beta-S (1 and 5 microg/g of body wt) was administered by intraperitoneal (ip) injection, 24 h prior to injection with LHRHa (10-50 ng/g of body wt) and fish were bled 1 h after LHRHa injection. Treatment with both doses of 20beta-S resulted in plasma concentrations of the steroid within the normal physiological range for this species during final oocyte maturation and ovulation. The 20beta-S treatments altered the LH response to LHRHa throughout the reproductive cycle in both sexes, but the direction and magnitude of the response varied. 20beta-S treatment decreased the LH response to LHRHa in fish with recrudescing and fully recrudesced gonads and in females with regressed gonads. On the other hand, 20beta-S treatment significantly increased the LH response to LHRHa in males with regressing or regressed gonads. 20beta-S treatment also altered preoptic anterior hypothalamic (POAH) and pituitary seabream gonadotropin-releasing hormone (sbGnRH) contents, and the patterns of these changes were similar to those observed in LH secretion. The finding that moderate increases in plasma 20beta-S concentrations, similar to those occurring during final oocyte maturation, significantly inhibit the LH response to LHRHa at the end of the reproductive cycle suggests that this action of 20beta-S is of physiological importance during the periovulatory period. Moreover, the fact that concurrent changes occur in POAH and pituitary sbGnRH contents suggests that the actions of 20beta-S on LH secretion are at least partly mediated via the GnRH system.

Topics: Animals; Cortodoxone; Female; Gonadotropin-Releasing Hormone; Gonads; Kinetics; Luteinizing Hormone; Male; Perciformes; Pituitary Gland; Preoptic Area; Reproduction

The jundiá Rhamdia quelen (Quoy and Gaimard) is a teleost species from the Siluridae family and is an important species for aquaculture in temperate and subtropical climates. Gonad and blood tissue samples were taken from cultured jundiá females between 1998 and 1999. Plasma concentrations of 17beta-estradiol (E(2)), testosterone (T), 11-ketotestosterone (11-KT), 17-hydroxy-4-pregnene-3,20-dione (17-P), 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P), and 17,20beta,21-trihydroxy-4-pregnen-3-one (20beta-S) were measured by radioimmunoassay and potential correlations with the stage of oogenesis and sexual maturation examined. During the experimental period two spawning episodes were observed. Plasma concentrations of E(2) increased progressively during oocyte development, simultaneously with the appearance of yolk vesicles and increasing amounts of deposited yolk. In female jundiá, the T peak occurred in October and was coincident with the peak in gonadosomatic index. Two distinct peaks of progestogens were detected, corresponding to the two spawning episodes, suggesting that one or more of these steroids might act as the "maturational-inducing steroid" in jundiá. Unusually large amounts of 11-KT were also measured in the plasma of mature jundiá females. The identity of 11-KT was confirmed by thin-layer chromatography. Although the profiles of the other steroids are compatible with the roles proposed for the action of these hormones in other teleosts, the role of 11-KT, normally found only in males, is unknown.

Topics: Animals; Climate; Cortodoxone; Estradiol; Female; Fishes; Hydroxyprogesterones; Oogenesis; Pregnenediones; Reproduction; Seasons; Sexual Maturation; Steroids; Testosterone

The aim of this study was to identify the major C21 steroids produced in vivo during artificially induced final oocyte maturation and spawning in female common dentex (Dentex dentex). During the spawning season, mature females were treated with a gonadotropin-releasing hormone agonist (GnRHa)-loaded delivery system, with or without pimozide (given as a single dose at the beginning of the experiment). Blood samples were collected at various intervals during the experiment and were assayed for GnRHa, 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P), and 17,20beta,21-trihydroxy-4-pregnen-3-one (17,20beta,21-P). A higher percentage of ovulated females was observed in GnRHa-implanted fish, which produced over 10 times more eggs than controls. Relative fecundity was highest in the GnRHa + pimozide group and lowest in controls. The viability of naturally released eggs was low (2 to 15%) in all groups. Plasma concentrations of 17,20beta-P in GnRHa-implanted fish did not increase, but those in control fish decreased, such that there was a significant difference between control and treated fish between 2 and 10 days after treatment. In another experiment, ovulating common dentex were injected intramuscularly with a single dose of 50 microg kg(-1) of GnRHa in saline and were sampled for blood at 0, 3, 6, 12, and 24 h postinjection. A single water sample was taken from the tanks at 9 h postinjection, the tanks having been emptied and refilled at 6 h. Measurements were made of plasma and water concentrations of free and conjugated 17,20beta-P, 17,20beta,21-P, 17beta-oestradiol (E2), and GnRHa (plasma only). The GnRHa injection increased plasma levels of all steroids, with free 17,20beta-P reaching maximal levels within 3 h. GnRHa treatment also increased the amounts of free and conjugated steroids released into the water between 6 and 9 h.

Topics: Animals; Cortodoxone; Drug Implants; Estradiol; Female; Fishes; Glucuronides; Gonadotropin-Releasing Hormone; Hydroxyprogesterones; Kinetics; Ovulation; Pimozide; Radioimmunoassay; Steroids; Sulfates; Water

A previous ultrastructural study of heterologous (granulosa cell-oocyte) gap junction (GJ) contacts in ovarian follicles of Atlantic croaker suggested that these contacts disappear late during the process of resumption of oocyte meiosis. This observation suggested that, unlike scenarios proposed for a number of other species, uncoupling of GJ is not necessary for the onset of meiotic resumption in croaker follicles. However, the functionality of heterologous GJ contacts and the temporal association between maturation-inducing hormone (MIH)-induced changes in heterologous coupling and resumption of oocyte meiosis have not been examined in Atlantic croaker. These questions were addressed with a cell-cell coupling assay that is based on the transfer of a GJ marker, Lucifer Yellow, from oocytes to granulosa cells. Follicle-enclosed oocytes injected with Lucifer Yellow allowed transfer of the dye into the follicle cell layer, thus confirming that there is functional heterologous coupling between the oocyte and the granulosa cells. Dye transfer was observed in vitellogenic, full-grown/maturation-incompetent, and full-grown/maturation-competent follicles. Treatment of maturation-competent follicles with MIH caused a time-dependent decline in the number of follicles transferring dye. However, although GJ uncoupling in some of the follicles was observed before germinal vesicle breakdown (GVBD, index of meiotic resumption), about 50% of the follicles maintained the ability to transfer dye even after GVBD had occurred. Further, a known GJ inhibitor (phorbol 12-myristate 13-acetate) blocked heterologous GJ within a time frame similar to that seen with MIH but without inducing any of the morphological changes (including GVBD) associated with follicular maturation. In conclusion, uncoupling of heterologous GJ seems insufficient and unnecessary for the onset of meiotic resumption in ovarian follicles of Atlantic croaker.

Topics: Animals; Cortodoxone; Female; Gap Junctions; Granulosa Cells; Oocytes; Ovarian Follicle; Perciformes

This study is the first investigation of reproductive endocrinology in a simultaneously hermaphroditic teleost, the belted sandfish (Serranus subligarius). We address two questions: (1) Do steroid hormone levels vary during the spawning season or during the daily spawning cycle of sandfish? (2) Do hormone levels vary relative to an individual's phenotype-size, frequency of spawning and aggressive behaviors, and proportion of testis in the gonad? We analyzed circulating estradiol-17beta (E2), testosterone (T), 11-ketotestosterone (11KT), 17alpha,20beta,21-trihydroxy-4-pregnen-3-one (20betaS), and 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP) concentrations in a field population. Only E2 levels were significantly higher at the new and full moon, suggesting peak periods of vitellogenesis at these times. Naturally spawning sandfish were sampled every 2 h during the photophase of a 25-h period (12 pm to 1 pm the following day) and gonadosomatic index, degree of oocyte hydration and ovulation, and plasma levels of E2, T, DHP, and 20betaS were analyzed. E2 and T levels did not vary during photophase, suggesting continuous recruitment of oocytes into vitellogenesis. The 20betaS levels peaked around the time of final oocyte maturation. Since frequency of spawning behaviors changes with body size, we captured individuals of various sizes throughout the spawning season and analyzed circulating levels of hormones. 11KT and 20betaS levels increased significantly with body size. In 1992, we quantified frequency of spawning and aggressive behaviors, circulating T and 11KT levels and testicular mass relative to ovotestis mass in focal animals. 11KT levels tended to be positively correlated with frequency of courting male behavior, but were unrelated to the frequency of aggressive behavior or testis mass. Because hormone levels increased with size and frequency of each spawning behavior changes with size, we propose that sex steroids influence growth-related changes in spawning tactics of individuals.

Topics: Aggression; Animals; Circadian Rhythm; Cortodoxone; Disorders of Sex Development; Estradiol; Female; Hydroxyprogesterones; Male; Oocytes; Organ Size; Perciformes; Regression Analysis; Seasons; Sexual Behavior, Animal; Testis; Testosterone

Recombinant human (rh) insulin-like growth factor-I (IGF-I) was more potent than rhIGF-II at inducing in vitro germinal vesicle breakdown (GVBD), a marker for resumption of meiosis, in oocytes of striped bass. Treatment of ovarian fragments containing oocytes in intact follicles with rhIGF-I increased concentrations of estradiol-17beta and maturation-inducing steroid (MIS) 17,20beta, 21-trihydoxy-4-pregnen-3-one (20beta-S) in the culture medium and decreased testosterone levels. The follicles were too immature for oocytes to complete GVBD in response to 20beta-S (MIS incompetent) or hCG. Addition of 20beta-S to cultures did not increase the percentage of oocytes completing GVBD in response to rhIGF-I or rhIGF-II. Bovine insulin was without effect on GVBD or steroid production. Incubation of MIS-competent follicles with actinomycin D, cyanoketone, trilostane, 1-heptanol, or 1-octanol had no effect on rhIGF-I-induced GVBD, but attenuated hCG-induced GVBD and 20beta-S production. Cycloheximide inhibited rhIGF-I-induced GVBD. Collectively, these observations indicate that IGF-I can induce GVBD via MIS- and transcription-independent pathways without coupled gap junctions between oocytes and granulosa cells or among granulosa cells, but requires protein synthesis to do so. An rhIGF-I analogue that does not bind IGF-binding proteins, des(1,3)IGF-I, was more potent than rhIGF-I in inducing GVBD, suggesting ovarian IGF-binding proteins may inhibit IGF-I action.

Topics: 1-Octanol; Animals; Bass; Cattle; Chorionic Gonadotropin; Cortodoxone; Cyanoketone; Cycloheximide; Dactinomycin; Dihydrotestosterone; Female; Heptanol; Humans; Hydroxysteroid Dehydrogenases; In Vitro Techniques; Insulin; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Oocytes; Ovary; Peptide Fragments; Recombinant Proteins; Steroids; Transport Vesicles; Uncoupling Agents

Characteristics of the lunar reproductive cycle in the golden rabbitfish, Siganus guttatus, were determined by histological observations of ovarian development, and immunological measurements of plasma steroid hormones, estradiol-17beta (E2), testosterone (T), 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP) and 17alpha,20beta,21-trihydroxy-4-pregnen-3-one (20beta-S), and vitellogenin (VTG). Ovarian and plasma samples were collected every week according to the lunar phases from May to July. Weekly change of gonadosomatic index (GSI) showed two peaks at the first lunar quarter in June and July. Yolky oocytes were also observed around this time. Histological observations revealed that the vitellogenic oocytes appeared again 1 week after spawning and developed synchronously. These results suggest that this species is a multiple spawner and the oocyte development is in a group-synchronous manner. Plasma steroid hormones (E2, T, DHP and 20beta-S) and VTG levels changed in parallel with changes in GSI. The peak of plasma VTG level occurred prior to spawning. These cyclic changes of plasma steroid hormones and VTG support the hypothesis that lunar periodicity is the major factor in stimulating reproductive activity of S. guttatus.

Topics: Animals; Cortodoxone; Estradiol; Female; Hydroxyprogesterones; Moon; Optical Illusions; Ovary; Perciformes; Seasons; Steroids; Testosterone; Time Factors; Vitellogenins

Although many environmental contaminants disrupt endocrine function by binding to nuclear steroid receptors, it is not known whether they are capable of binding to steroid membrane receptors and interfering with nongenomic actions of steroids. The binding of several organochlorine pesticides to the plasma membrane receptor for the maturation-inducing steroid, 17,20beta,21-trihydroxy-4-pregnen-3-one (20beta-S), in the ovaries of spotted seatrout (Cynoscion nebulosus) was investigated in in vitro competition assays. Kepone and o,p'-DDD were competitive inhibitors of 20beta-S binding and caused concentration-dependent displacement of [3H]-20beta-S from its receptor site over the range of 10(-4) to 10(-6) or 10(-7) M, whereas several other pesticides had lower affinities for the receptor. Interference with the nongenomic actions of 20beta-S on final meiotic maturation of spotted seatrout oocytes (final oocyte maturation, FOM) was examined in an in vitro bioassay. A concentration-dependent inhibition of FOM in response to 20beta-S was observed after 5 min and 12 h exposure to the same range of Kepone and o,p'-DDD concentrations (10(-4) to 10(-6) or 10(-7) M). The close correspondence between competitive binding of the two pesticides to the 20beta-S membrane receptor and their inhibition of 20beta-S induced FOM suggests a mechanism of endocrine disruption mediated by binding to a steroid membrane receptor and antagonism of a nongenomic steroid action.

Topics: Animals; Binding, Competitive; Cell Membrane; Chlordecone; Cortodoxone; DDT; Female; Insecticides; Meiosis; Methoxychlor; Mitotane; Oocytes; Perciformes; Receptors, Progesterone

Incubation of mature, hydrated, follicle-enclosed oocytes of the spotted seatrout, Cynoscion nebulosus, with the maturation-inducing steroid (MIS), 17,20beta,21-trihydroxy-4-pregnen-3-one (20beta-S), for 9-12 h resulted in the appearance of ovulated oocytes in the culture media. The ovulation response was concentration-dependent and steroid-specific. The other teleost MIS, 17, 20beta-dihydroxy-4-pregnen-3-one (17,20beta-P), was also a potent inducer of ovulation, whereas progesterone and 11-deoxycorticosterone did not stimulate ovulation above control levels and partially antagonized the action of 20beta-S. The agonist and antagonist activities of these steroids on ovulation are consistent with their relative binding affinities for the ovarian nuclear progestogen receptor previously characterized in this species. Both the RNA synthesis inhibitor actinomycin D and the protein synthesis inhibitor cycloheximide blocked MIS-induced ovulation. This suggests that induction of ovulation by the MIS is through a genomic mechanism of action, and potentially involves the previously characterized nuclear progestogen receptor. Gonadotropin (hCG)-induced ovulation was blocked by addition of the steroid synthesis inhibitor cyanoketone, which was overcome by the addition of 20beta-S, but not pregnenolone. Thus, the most likely mechanism of gonadotropin-induced ovulation is an increase in the synthesis of the MIS. It is concluded that the processes of final oocyte maturation and ovulation are both regulated by the MIS. Whereas final oocyte maturation is mediated by the 20beta-S membrane receptor (P. Thomas and S. Das, 1997, Biol. Reprod. 57, 999-1007), ovulation is regulated by a genomic mechanism and is potentially mediated by the previously characterized nuclear progestogen receptor.

Topics: Animals; Chorionic Gonadotropin; Cortodoxone; Female; Oocytes; Ovary; Ovulation; Progestins; Receptors, Cytoplasmic and Nuclear; Receptors, Progesterone; Trout

Previous studies have shown that blood plasma levels of 17alpha, 20beta-dihydroxy-4-pregnen-3-one (DHP) and 17alpha, 20beta, 21-trihydroxy-4-pregnen-3-one (20beta-S) increase in striped bass (Morone saxatilis) undergoing final oocyte maturation (FOM). Both hormones are produced by ovarian fragments undergoing hCG-induced germinal vesicle breakdown (GVBD) in vitro. In the present study, we investigated binding of DHP and 20beta-S to ovarian membranes from striped bass undergoing FOM. Saturable binding sites for DHP were not detected. Saturation of 20beta-S binding sites with 5 nM [3H]20beta-S occurred within 40 min at 0 degrees C (at 3 min, half of the maximum specific binding of steroid was calculated to have occurred), and the binding was pH-dependent. Scatchard analyses revealed the presence of a single class of high-affinity (dissociation constant [Kd] = 1.4 +/- 0.2 nM), limited-capacity (estimated concentration [Bmax] = 2.7 +/- 0.3 pmol/g ovary) 20beta-S binding sites on membranes from striped bass ovaries undergoing FOM. In contrast, only low levels of specific binding (Bmax < 0.04 pmol/g tissue) were detected on membranes from testes, liver, brain, and muscle. Ovarian membranes prepared from vitellogenic females also had low levels (Bmax < 0.1 pmol/g ovary) of specific 20beta-S binding, less than 5% of that found during FOM. Results of competition assays showed that DHP was approximately 250 times less effective than 20beta-S for displacing 20beta-S from ovarian membranes. In contrast, 20beta, 21-dihydroxy-4-pregnen-3-one was a very effective competitor, although it is only a weak inducer of oocyte GVBD in vitro. Of several other steroids tested, only progesterone showed affinity for the 20beta-S binding site within a physiological range of concentrations. Taken together with previous studies of striped bass FOM, these findings indicate that 20beta-S is the oocyte maturation-inducing steroid hormone in striped bass.

Topics: Animals; Bass; Binding, Competitive; Cell Membrane; Cortodoxone; Female; Hydrogen-Ion Concentration; Hydroxyprogesterones; Kinetics; Oocytes; Ovary; Receptors, Cell Surface

Ovaries from female turbot (Scophthalmus maximus L.), a serial spawner, were incubated in vitro with 17-hydroxy[1,2,6,7-3H]progesterone or [7-3H(N)]pregnenolone (P5-3H) during the spawning season. Several metabolites comigrated on TLC and HPLC with known reference steroids and were identified after chemical reaction and crystallization. Incubation with P5-3H generated many 4-ene steroids, accounting for 55% of total radioactivity, indicating strong 3 beta-HSD activity. The major steroids produced by ovaries were testosterone, androstenedione, and 17,21-dihydroxy-4-pregnene-3,20-dione. In addition, 17,20 alpha-dihydroxy-4-pregnen-3-one was also identified, while small quantities of 17,20 beta,21-trihydroxy-4-pregnen-3-one (20 beta-S) (maximum 1.7% of the total radioactivity) were also synthesized. The identity of 20 beta-S was confirmed in incubates with nonlabeled 17-hydroxyprogesterone by mass spectrometry. Production of 17,20 beta-dihydroxy-4-pregnen-3-one was not apparent.

Topics: 17-alpha-Hydroxyprogesterone; Animals; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Cortodoxone; Female; Flatfishes; In Vitro Techniques; Mass Spectrometry; Oocytes; Ovary; Pregnenolone; Sexual Maturation

Spermiating striped bass, a perciform fish, were treated with two controlled-release gonadotropin-releasing hormone agonist (GnRHa)-delivery systems, and the resulting changes in plasma gonadotropin II (GTH II), testosterone (T), 11-ketotestosterone (11-KT), 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P), and 17,20beta,21-trihydroxy-4-pregnen-3-one (17,20beta,21-P) were correlated with changes in milt production and sperm density. GnRHa-delivery systems induced a sustained elevation of plasma GnRHa and GTH II for 14 days. Plasma T levels were unchanged after GnRHa treatment, while 11-KT levels increased significantly. Plasma 17,20beta-P also increased after GnRHa treatment and remained elevated compared to levels in controls, while plasma 17,20beta,21-P levels were unaffected. Both GnRHa-delivery systems induced many-fold increases in total expressible milt, lasting throughout the 14 days of the study. Sperm density decreased 2 days after GnRHa treatment, with a subsequent increase by Day 7. This study demonstrates that GnRHa-delivery systems induce a sustained elevation of plasma GTH II levels in striped bass, resulting in a long-term enhancement of milt production. The endocrine changes observed suggest that 11-KT and 17,20beta-P, but not 17,20beta,21 -P, are mediating the effects of GTH II on spermiation.

Topics: Animals; Bass; Cortodoxone; Drug Delivery Systems; Gonadal Steroid Hormones; Gonadotropin-Releasing Hormone; Gonadotropins, Pituitary; Hydroxyprogesterones; Male; Spermatogenesis; Testosterone

Female and male tilapia, Oreochromis mossambicus, were treated with luteinizing hormone-releasing hormone analogue (LH-RHa) and pimozide (PIM) or with human chorionic gonadotropin (hCG) to stimulate gonadal development and sexual maturation. Plasma (both sexes) and urine (males) samples were collected periodically for steroid analysis by radioimmunoassay. Plasma levels of estradiol-17 beta (3-6 ng/ml) and testosterone, higher in female (up to 25 ng/ml) than in male (6-13 ng/ml; P < 0.05), were in the range of those established in other tilapia species. Plasma levels of the established teleost oocyte maturation-inducing steroids (MIS), that is 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-P) and 17 alpha,20 beta, 21-trihydroxy-4-pregnen-3-one (17,20 beta,21-P) were low (1-9 ng/ml) and were not different between treated and control fishes at 8, 12, 16, 24, 48, 72 and 96 hr after injection. Furthermore, in male O. mossambicus, 17,20 beta,21-P was undetectable. Plasma levels of 3 alpha,17 alpha,21-trihydroxy-5 beta-pregnan-20-one (3,17,21-P-5 beta) were very high in both sexes (up to 700 ng/ ml), mostly in hormone-treated groups, whose levels were higher than controls (P < 0.05). Urine levels of conjugated 17,20 beta,21-P (glucuronides and sulphates) were not detectable, but those of 17, 20 beta-P (up to 25 ng/ ml) and 3,17,21-P-5 beta (up to 1 microgram/ml) were higher than free 17,20 beta-P and 3,17,21-P-5 beta measured in the plasma of the same animals (P < 0.05). Both LH-RHa + PIM and hCG induced sexual maturation of O. mossambicus (histological data); nevertheless, during that period all measured steroids, either in plasma or urine, almost did not fluctuate. Thus, this study does not make any comment about the MIS of tilapia. Nevertheless, the high levels of conjugated 3,17,21-P-5 beta and 17,20 beta-P in urine suggest a probable pheromone role for these steroids in this species.

Topics: Animals; Chorionic Gonadotropin; Cortodoxone; Estradiol; Female; Gonadotropin-Releasing Hormone; Gonads; Humans; Hydroxyprogesterones; Male; Pimozide; Radioimmunoassay; Sexual Maturation; Steroids; Testosterone; Tilapia; Time Factors

Levels of estradiol-17 beta (E2), testosterone (T), 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (DHP), and 17 alpha,20 beta,21-trihydroxy-4-pregnen-3-one (20 beta-S) were measured by radioimmunoassay (RIA) in blood plasma of striped bass undergoing final oocyte maturation (FOM). Females were captured just prior to, or in the early stages of, FOM and induced to complete maturation and ovulation with injected human chorionic gonadotropin, synthetic salmon gonadotropin-releasing hormone analogue (sGnRHa; [D-Arg6-Pro9 NEt]-sGnRH), sGnRHa plus the dopamine receptor antagonist, domperidone (DOM), or OVAPRIM, a commercial preparation of sGnRHa + DOM. Their plasma levels of immunoreactive DHP and 20 beta-S were significantly greater at ovulation relative to the time of hormone injection, whereas the plasma levels of E2 and T were greatest at injection and decreased by ovulation and 24 hr thereafter. Plasma levels of 20 beta-S, but not DHP, were sustained at high levels after ovulation. Fish injected only with DOM did not undergo FOM, its associated changes in plasma steroid levels, or ovulation. In females captured at various natural stages of FOM, plasma levels of 20 beta-S and DHP were low during germinal vesicle migration (GVM), peaked coincident with germinal vesicle breakdown, and then decreased near the time of ovulation. Plasma levels of E2 and T were greatest during GVM and decreased as DHP and 20 beta-S levels increased. Analyses of conjugated versus free plasma steroids showed 64-79% of the various hormones to be in the free fraction. RIA of plasma fractionated by reversed-phase HPLC showed that half of the 20 beta-S immunoreactivity coeluted with 5 beta-pregnan-3 alpha,17,20 beta,21-tetrol, a putative 20 beta-S metabolite with 99.7% cross-reactivity in the 20 beta-S RIA. These results indicate that striped bass follow the typical profile of changing plasma steroid levels seen in other teleosts during FOM, with a clear shift from C18 and C19 steroids to C21 steroids. They suggest that both DHP and 20 beta-S, both potent inducers of striped bass FOM in vitro, may play a role in regulating FOM in this species.

Topics: Animals; Bass; Chorionic Gonadotropin; Cortodoxone; Domperidone; Estradiol; Female; Gonadal Steroid Hormones; Gonadotropins; Hydroxyprogesterones; Oocytes; Ovulation; Radioimmunoassay; Reproduction; Testosterone

Full-grown oocytes of Atlantic croaker are insensitive to maturation-inducing steroid (MIS) unless they are primed with gonadotropin (GtH). The objective of this study was to examine the mechanism of GtH-induced maturational competence in croaker oocytes. Specifically, we determined the in vitro secretion of steroids by intact ovarian follicles of unprimed or hCG-primed fish, the direct effects of steroids on maturational competence, and the effects of steroid (cyanoketone), protein (cycloheximide), and RNA (actinomycin D) synthesis inhibitors on hCG-induced maturational competence and steroidogenesis in vitro. The steroid content of the incubation medium after hCG treatment was measured by RIA. The effects of hCG or exogenous steroid treatment on maturational competence were determined by recording the incidence of germinal vesicle breakdown (GVBD) after MIS-induced GVBD in a standard bioassay. Our major findings were: (1) induction of maturational competence occurred after exposure of ovarian follicles to hCG either in vivo or in vitro; (2) MIS secretion was detected in follicles of hCG-primed fish but not unprimed fish, and no MIS secretion was observed during hCG induction of maturational competence in vitro; (3) treatment with cyanoketone blocked the hCG-dependent secretion of testosterone and estradiol but not the development of maturational competence; (4) treatment with MIS or various other exogenous steroids in the absence of hCG did not induce maturational competence; and (5) hCG-induced maturational competence was inhibited by cycloheximide and actinomycin D. Therefore, the mechanisms of GtH induction of oocyte maturation in Atlantic croaker can be described in two distinct stages: a delta-4 steroid-(including MIS) and estrogen-independent priming stage followed by a MIS-mediated GVBD stage. The priming stage may involve mechanisms requiring RNA as well as protein synthesis.

Topics: Animals; Chorionic Gonadotropin; Cortodoxone; Cyanoketone; Cycloheximide; Dactinomycin; Estradiol; Female; Gonadotropins; Oocytes; Oogenesis; Ovary; Perciformes; Radioimmunoassay; Testosterone; Time Factors

The proposed maturation-inducing substance (MIS) of spotted seatrout (Cynoscion nebulosus) is 17 alpha, 20 beta, 21-trihydroxy-4-pregnen-3-one (20 beta-S). In this study, we characterized the binding of radioactive 20 beta-S to plasma membranes from the ovaries of spotted seatrout. Bound 20 beta-S was isolated by filtration of membrane suspensions and quantified by measurement of the radioactivity content of the filters. The saturable component of 20 beta-S binding reached equilibrium within 5 min at 0 degree, showed linearity with membrane concentration, and was pH dependent (optimum, 7.5-7.8). Scatchard analyses suggested a single class of high-affinity (KD, 10(-9) M), low-capacity (10(-13)-10(-12) mol/g ovary) binding sites for 20 beta-S. High levels of saturable binding were found in membrane preparations from the ovary, testis, and liver, but not from the gills. 17 alpha, 20 beta-Dihydroxy-4-pregnen-3-one (17 alpha, 20 beta-P) showed relatively little affinity for the 20 beta-S binding site. However, this steroid was converted to a compound immunologically and chromatographically similar to 20 beta-S by intact ovarian follicles, a finding which may explain its previously reported high potency in an in vitro oocyte maturation bioassay. Conversely, although reportedly a weak inducer of oocyte maturation, progesterone readily displaced 20 beta-S from its binding site. Thus, progesterone appears to be a relatively inactive ligand with high affinity for the 20 beta-S receptor. The concentration of 20 beta-S binding sites in ovaries was significantly higher during final oocyte maturation (germinal vesicle migration) than at earlier stages of development. These results strongly suggest that the 20 beta-S binding activity characterized in our study represents authentic MIS receptors. A distinct, soluble binding site for 17 alpha, 20 beta-P was also identified in seatrout ovaries, but its biological function remains unclear. A hypothesis is presented for the significance of this 17 alpha,20 beta-P binding site.

Topics: 17-Hydroxycorticosteroids; Animals; Cell Membrane; Cortodoxone; Female; Gills; Hydrogen-Ion Concentration; Liver; Male; Ovary; Radioimmunoassay; Receptors, Steroid; Salmonidae; Testis; Trout

We characterized the in vitro control of germinal vesicle breakdown (GVBD) by 17 alpha,20 beta,21-trihydroxy-4-pregnen-3-one (20 beta-S) in intact ovarian follicles of gonadotropin-primed Atlantic croaker. 20 beta-S-induced GVBD was determined in relation to ovarian (oocyte) morphology, duration of incubation, steroid metabolism, and interaction with other steroids. The rate of GVBD in vitro in the absence of exogenous steroid was positively correlated with initial stage of ovarian morphological development. Maximal responsiveness to 20 beta-S was seen in ovaries with oocytes showing the first signs of morphological maturation. Dose-response experiments with 20 beta-S and 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-P) over a range of incubation times yielded similar results for both steroids, suggesting that conversion of 17 alpha,20 beta-P to 20 beta-S is not required for 17 alpha,20 beta-P-induced GVBD. The ED50 of these steroids markedly decreased with increasing incubation times. Comparisons between patterns of follicular transformation of various radiolabelled steroids to 20 beta-S and their respective activities (using unlabelled steroids) in the GVBD bioassay suggested that, in addition to 17 alpha,20 beta-P, progesterone has some intrinsic maturational activity. However, the maturational effects of 11-deoxycortisol and pregnenolone may be explained by their conversion to 20 beta-S. For the first time in any vertebrate, we showed that the proposed maturation-inducing steroid (20 beta-S) is not significantly transformed to any extractable, potentially active metabolite by intact, maturing ovarian follicles. These findings strongly suggest that 20 beta-S is the terminal product of the MIS biosynthetic pathway in Atlantic croaker ovaries. Estradiol had no acute effects on 20 beta-S-induced GVBD. However, testosterone decreased and cortisol augmented the maturational activity of 20 beta-S. Excess progesterone reduced the activity of a maximally effective dose of 20 beta-S, but pregnenolone was without effect. The effects of these steroids on 20 beta-S-induced GVBD are discussed in relation to their possible interactions with 20 beta-S at the MIS receptor level.

Topics: 17-Hydroxycorticosteroids; Adrenal Cortex Hormones; Analysis of Variance; Animals; Cortodoxone; Female; Fishes; Gonadal Steroid Hormones; Hydroxyprogesterones; In Vitro Techniques; Oogenesis; Ovarian Follicle; Steroids; Time Factors

Radioimmunoassays, combined with thin-layer chromatography, have been used to test for the presence of 17 alpha,20 beta-dihydroxy-4-pregnen-3-one, 17 alpha,20 beta,21-trihy-droxy-4-pregnen-3-one, and 20 beta-hydroxy-4-pregnen-3-one, and for the presence of their 5-pregnene and 5 alpha-pregnane analogs, in the blood and ovarian incubation media of maturing/ovulating female rainbow trout (Salmo gairdneri). All of these steroids have previously been shown to be more or less equipotent in in vitro oocyte final maturation assays. Only two steroids were found: 17 alpha,20 beta-dihydroxy-4-pregnen-3-one, the presence of which has already been firmly established, and 17 alpha,20 beta, 21-trihydroxy-4-pregnen-3-one, which has not been previously identified in rainbow trout. The amounts of 17 alpha,20 beta, 21-trihydroxy-4-pregnen-3-one found in vivo and in vitro were, however, only 1-1.5% of those of 17 alpha,20 beta-dihydroxy-4-pregnen-3-one, and therefore seem unlikely to play a significant role in the induction of oocyte final maturation.

Topics: 17-Hydroxycorticosteroids; 20-alpha-Dihydroprogesterone; Animals; Cortodoxone; Cross Reactions; Dose-Response Relationship, Drug; Female; Gonadotropins; Hydroxyprogesterones; Immune Sera; Oocytes; Ovary; Ovulation; Progesterone; Radioimmunoassay; Salmonidae; Trout

All the steroids produced by Atlantic croaker ovaries during final oocyte maturation (FOM) were assayed for their ability to induce germinal vesicle breakdown (GVBD) of croaker oocytes in vitro. Ovarian tissue in the process of FOM was removed from Atlantic croaker (Micropogonias undulatus) and incubated with human chorionic gonadotropin (hCG) and pregnenolone in tissue culture medium for 8 hr. Steroids were extracted from the medium and fractionated by HPLC and TLC. Fractions were bioassayed for their potency to induce GVBD of Atlantic croaker oocytes in vitro. 17 alpha, 20 beta,21-Trihydroxy-4-pregnen-3-one (20 beta-dihydro-11-deoxycortisol, 20 beta-S) was identified as the predominant steroid product and the major maturation-inducing steroid produced by the ovary of Atlantic croaker in vitro. A small amount of another steroid with equal potency to induce GVBD was tentatively identified as 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-P4); however, over ten times more 20 beta-S than 17 alpha,20 beta-P4 accumulated in the incubation medium. Trace amounts of testosterone and estradiol-17 beta were also isolated. Ovarian tissue incubated under more physiological conditions, without the addition of excess pregnenolone, failed to produce 17 alpha,20 beta-P4. These results provide further evidence that 20 beta-S is a major maturation-inducing steroid in Atlantic croaker.

Topics: 17-Hydroxycorticosteroids; Animals; Cortodoxone; Female; In Vitro Techniques; Oocytes; Ovary; Perciformes

Changes in ovarian steroidogenesis during final oocyte maturation (FOM) in the Atlantic croaker (Micropogonias undulatus) were investigated in vitro by incubation of oocytes with tritiated pregnenolone, followed by chromatographic separation of the radioactive steroid products. Testosterone and estradiol-17 beta were the major tritiated steroids produced by the ovary before the onset of FOM. Induction of FOM by human chorionic gonadotropin, both in vivo and in vitro, coincided with the appearance of 17 alpha,20 beta,21-trihydroxy-4-pregnen-3-one (20 beta-S) as the predominant steroid product. The production of 17 alpha,20 beta-dihydroxy-4-pregnen-3-one and 11-deoxycorticosterone, maturation-inducing steroids previously proposed for other teleost species, could not be demonstrated in Atlantic croaker ovaries during FOM. The timely synthesis of 20 beta-S in croaker at the onset of oocyte maturation provides further evidence for a physiological role of 20 beta-S in the induction of FOM in this species.

Topics: 17-Hydroxycorticosteroids; Animals; Cortodoxone; Female; Gonadal Steroid Hormones; In Vitro Techniques; Oocytes; Ovary; Perciformes

The present study investigated the effects of 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17,20 B-P) on (1) ovulation of isolated perch follicles in which the extrafollicular (EF) ovarian tissue had been removed; (2) prostaglandin F (PGF) and prostaglandin E (PGE) synthesis in EF tissue and intact (= follicles attached to EF tissue) and isolated follicles by radioimmunoassay and [14C]arachidonic acid incorporation; and (3) proteolysis in EF tissue and intact and isolated follicles by substrate sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition, the ovulatory and proteolytic effects of the phorbol ester, phorbol-12-myristate-13-acetate (PMA), and the calcium ionophore A23187 on 17,20B-P-stimulated follicles were also studied in the presence/absence of indomethacin (IM) and nordihydroguaiaretic acid (NDGA). Ultrastructural analyses revealed that the preparation of isolated follicles also removed the surface epithelium of the follicle. While 17,20B-P stimulated ovulation and an increase in PGF and PGE in incubates of intact perch follicles, it did not in incubates of isolated follicles. In contrast, PMA, A23187, and a combination of PMA and A23187 stimulated ovulation of these isolated follicles. PMA/A23187-induced ovulation could be blocked by NDGA but not IM, and two hydroxyeicosatetraenoic acids (11- and 15-HETEs) were capable of partially reversing the NDGA block. Incorporations with [14C]arachidonic acid revealed that the EF tissue had a significant potential to produce PGF; however, 17,20B-P did not stimulate an increase in PGF or PGE (measured by RIA) in incubates of EF tissue alone. In addition, neither ovulation nor an increase in prostaglandins was observed in cocultures of isolated follicles and EF tissue. One major protease (66 kDa) was observed in the medium during incubation of intact and isolated perch follicles. No protease activity was present in incubates of EF tissue alone. Protease activity in 17,20B-P-stimulated incubates of intact tissue was significantly higher than in steroid-stimulated incubates of isolated follicles. Protease activity increased in the medium during incubation with PMA or a combination of PMA and A23187. This activity was blocked by NDGA but not IM. The NDGA block was partially reversed by 11-HETE. The combined results suggest that there is an interaction of EF tissue and follicle that is necessary, particularly for the stimulation of ovulation and prostaglandin production. Further, the results wi

Topics: 17-Hydroxycorticosteroids; Animals; Cortodoxone; Female; In Vitro Techniques; Ovary; Ovulation; Perches; Perciformes; Phorbol Esters; Prostaglandins