THE CORNEAL ENDOTHELIUM IN OCULAR SURFACE DISEASE AND GLAUCOMA: MECHANISMS OF DYSFUNCTION AND TREATMENT STRATEGIES.

To determine risk factors and the overall incidence of ocular surface disorders in a cohort of long-term glaucoma patients. Utilizing simple clinical tools, cross-sectional observational research were constructed to evaluate ocular surface problems and indicators. Using a four-grade scale, ten queries regarding symptoms and indications on the cornea's surface were used to create an OSD severity score. The patients were divided into three groups: A, B, and C, depending on the result. The variables that increase the incidence of surface sickness were identified using a multinomial logistic regression. Five hundred and twenty patients made up the total population. According to the multivariate analysis, the patient's age, the number of daily eyedrops, any previous changes in topical treatment for ocular intolerance, intraocular pressure, and degree of glaucoma were all connected with the severity of ocular surface illness. Ocular surface disorders are frequently developed by patients getting treatment for primary open-angle glaucoma or ocular hypotension. which are less prevalent and serious in geriatric patients because their use greater drugs and have greater advanced glaucoma.

INNOVATIONS IN ARTIFICIAL ORGANS AND TISSUE ENGINEERING: FROM 3D PRINTING TO STEM CELL THERAPY.

"Every year, many individuals with tissue or organ problems require urgent care due to medical emergencies, burns, congenital anomalies, and other causes". Regenerative medicine was created because there aren't enough donors, issues with graft rejection, and insufficient organs or tissues for patients to replace, repair, and regenerate. However, significant tissue defects are difficult to fill with injections alone, making stem cell therapy a crucial component of the area of regenerative medicine. To achieve the intended outcome, the researchers combine stem cells with three-dimensional (3D) printed organs tissue engineering scaffolding. These scaffolds can resemble bone, cartilage, or "extracellular matrix (ECM)" in that they provide structural support and promote adhesion, proliferation, and differentiation, finally resulting in the production of functional tissues or organs. In this study on stem cell regenerative medicine, the therapeutic focused mostly on scaffolding for 3D printed organ tissue engineering. The following applications are demonstrated and compared using various 3D printing processes and starting materials. Then, we go over the benefits of 3D printing over conventional methods, touch on certain issues and restrictions, and make some assumptions about potential applications in the future.

PSORIASIS PATHOGENESIS: INSIGHTS FROM TRANSCRIPTOMICS AND PROTEOMICS STUDIES OF KERATINOCYTES.

Complex inflammatory skin disease with autoimmune roots is psoriasis. This disease affects various cell types, and the underlying signaling processes are complicated yet not fully understood. Extensive psoriatic lesions' proteome and transcriptome of several researches were combined to understand disease's underlying biological mechanisms. According to a network-based study, both transcriptomics and proteomics control were comparable. They discovered many pathways of signaling previously undiscovered and possibly involved in overexpression of psoriasis genes. They also found a collection of transcription factors in charge of this process. The functional overlap between the results of transcriptomics and proteomics was also examined. There created a network-based method for combining the analysis of many high-throughput data sources. Proteomic and transcriptome studies of psoriasis data sets demonstrated regulatory flexibility apparatus underpinning disease and complementary relationships within two cellular organizations.

Oncologic safety of submental island flap reconstruction in clinically node-negative oral cancer patients: a prospective comparative study.

The purpose of this study was to evaluate the oncologic safety of submental island flap (SIF) reconstruction in clinically node-negative oral cancer patients. Forty-four clinically node-negative oral cancer patients with tumour size T1-T3 were divided into two groups. The Submental group consisted of 21 patients, who underwent submental island flap reconstruction whereas the control group consisted of 23 patients who underwent reconstruction with other locoregional or free flaps. The locoregional recurrence rate (LRR) and recurrence-free survival (RFS) in these two groups were assessed and compared. The follow-up period in the two groups ranged from six to 28 months, with a median follow-up period of 15 months and 21 months, respectively. Results showed that the LRR in the control and the submental group was 21.7% and 19%, respectively (p = 0.825). Kaplan-Meier curve showed that the difference in recurrence-free survival in the two groups was not statistically significant (p = 0.749). Multivariate and bivariate analyses did not establish any relationship between the predictive parameters and locoregional recurrence. Thus, the Submental island flap is a reliable and versatile locoregional flap for the reconstruction of post-resection defects in oral cancer. It has no predictive influence on locoregional recurrence in clinically node-negative oral cancer patients.

Acute engagement of Gq-mediated signaling in the bed nucleus of the stria terminalis induces anxiety-like behavior.

The bed nucleus of the stria terminalis (BNST) is a brain region important for regulating anxiety-related behavior in both humans and rodents. Here we used a chemogenetic strategy to investigate how engagement of G protein-coupled receptor (GPCR) signaling cascades in genetically defined GABAergic BNST neurons modulates anxiety-related behavior and downstream circuit function. We saw that stimulation of vesicular γ-aminobutyric acid (GABA) transporter (VGAT)-expressing BNST neurons using hM3Dq, but neither hM4Di nor rM3Ds designer receptors exclusively activated by a designer drug (DREADD), promotes anxiety-like behavior. Further, we identified that activation of hM3Dq receptors in BNST VGAT neurons can induce a long-term depression-like state of glutamatergic synaptic transmission, indicating DREADD-induced changes in synaptic plasticity. Further, we used DREADD-assisted metabolic mapping to profile brain-wide network activity following activation of Gq-mediated signaling in BNST VGAT neurons and saw increased activity within ventral midbrain structures, including the ventral tegmental area and hindbrain structures such as the locus coeruleus and parabrachial nucleus. These results highlight that Gq-mediated signaling in BNST VGAT neurons can drive downstream network activity that correlates with anxiety-like behavior and points to the importance of identifying endogenous GPCRs within genetically defined cell populations. We next used a microfluidics approach to profile the receptorome of single BNST VGAT neurons. This approach yielded multiple Gq-coupled receptors that are associated with anxiety-like behavior and several potential novel candidates for regulation of anxiety-like behavior. From this, we identified that stimulation of the Gq-coupled receptor 5-HT2CR in the BNST is sufficient to elevate anxiety-like behavior in an acoustic startle task. Together, these results provide a novel profile of receptors within genetically defined BNST VGAT neurons that may serve as therapeutic targets for regulating anxiety states and provide a blueprint for examining how G-protein-mediated signaling in a genetically defined cell type can be used to assess behavior and brain-wide circuit function.

Bayesian geostatistical modelling with informative sampling locations.

We consider geostatistical models that allow the locations at which data are collected to be informative about the outcomes. A Bayesian approach is proposed, which models the locations using a log Gaussian Cox process, while modelling the outcomes conditionally on the locations as Gaussian with a Gaussian process spatial random effect and adjustment for the location intensity process. We prove posterior propriety under an improper prior on the parameter controlling the degree of informative sampling, demonstrating that the data are informative. In addition, we show that the density of the locations and mean function of the outcome process can be estimated consistently under mild assumptions. The methods show significant evidence of informative sampling when applied to ozone data over Eastern U.S.A.

Expression and localization of the CDC34 ubiquitin-conjugating enzyme in pediatric acute lymphoblastic leukemia.

Ubiquitin-dependent protein degradation impacts many cellular processes.However, the regulation of ubiquitin-conjugating enzymes (UBCs) in cancer is unknown. We find that the human CDC34 UBC protein is expressed at a 3-4 fold higher level (P < 0.001) in pediatric T cell than in pre-B-cell acute lymphoblastic leukemia (ALL) before treatment in two independent patient sets. The level of CDC34 mRNA was similar in both types of leukemia. CDC34 expression levels in normal resting T cells, B cells and activated T lymphocytes was comparable with pre-B-cell ALL. CDC34 protein (but not mRNA) was also increased in T-cell ALL compared with pre-B-cell ALL cell lines. The difference in expression was not attributable to mutation or associated with altered CDC34 stability. Immunohistochemistry and cellular fractionation reveals a heterogeneous CDC34 expression pattern including cells containing primarily cytoplasmic or nuclear protein. Thus, a feature of pediatric T-cell ALL is posttranscriptional up-regulation and heterogeneous localization of the human CDC34 UBC.

Direct action of GnRH variants on goldfish oocyte meiosis and follicular steroidogenesis.

This study investigated the effects of gonadotropin-releasing hormones (GnRH) present in the goldfish brain (sGnRH and cGnRH-II) as well as a number of other GnRH variants on the reinitiation of meiosis and testosterone production in the follicle-enclosed goldfish oocytes, in vitro. All GnRH peptides tested individually stimulated oocyte meiosis in vitro, as determined by germinal vesicle breakdown (GVBD) as well as histone H1 kinase activity, which is an indicator of maturation promoting factor (MPF) production. The GnRH peptides tested had no detectable effect on basal follicular testosterone production with the exception of lGnRH-III, which had a relatively small stimulatory effect. In the presence of gonadotropin hormone (GTH), however, both sGnRH and lGnRH-III inhibited GTH-induced meiosis and steroidogenesis, whereas other GnRH peptides had no effect on GTH-induced responses. Addition of a GnRH antagonist effectively blocked the stimulatory effect of all GnRH peptides on oocyte meiosis, but was without effect on the inhibitory actions of sGnRH and lGnRH-III on GTH-induced meiosis, suggesting the involvement of different pathways mediating the stimulatory and inhibitory actions of sGnRH and lGnRH-III. These GnRH peptides were found to bind to the GnRH receptors in the goldfish ovary with different affinities (equilibrium association constant, K(a)). The findings provide novel information on the activity of GnRH variants in the goldfish ovary and provide a strong support for the hypothesis that GnRH plays a paracrine/autocrine role in the regulation of ovarian function in goldfish.

Time-related effect of GnRH on histone H1 kinase activity in the goldfish follicle-enclosed oocyte.

The level of cyclin B-associated cdc2 kinase, a component of maturation promoting factor (MPF), is known to be high during metaphase of the meiotic maturation of oocytes. The time-related action of gonadotropin-releasing hormones (GnRH) on histone H1 kinase activity (known to reflect cdc2 kinase activity) was investigated in vitro in follicle-enclosed goldfish oocytes. Germinal vesicle breakdown (GVBD) and testosterone production were also investigated in the same follicle-enclosed goldfish oocytes to determine the temporal relationship between GnRH-induced histone H1 kinase activity and the reinitiation of meiosis and steroidogenesis. Treatments with gonadotropin (GTH) or GnRH stimulated the histone H1 kinase activity to the same maximum level. However, sGnRH- and cGnRH-II-induced histone H1 kinase activity were significantly higher compared with controls after 2 hours of treatment, whereas the GTH-induced increase became significantly higher after 6-8 hours of incubation. Overall, the results demonstrate a close temporal relationship between GVBD response and histone H1 kinase activity induced by GTH and sGnRH-cGnRH-II.

Human Cdc34 and Rad6B ubiquitin-conjugating enzymes target repressors of cyclic AMP-induced transcription for proteolysis.

Ubiquitin-mediated proteolysis controls diverse physiological processes in eukaryotes. However, few in vivo targets of the mammalian Cdc34 and Rad6 ubiquitin-conjugating enzymes are known. A yeast-based genetic assay to identify proteins that interact with human Cdc34 resulted in three cDNAs encoding bZIP DNA binding motifs. Two of these interactants are repressors of cyclic AMP (cAMP)-induced transcription: hICERIIgamma, a product of the CREM gene, and hATF5, a novel ATF homolog. Transfection assays with mammalian cells demonstrate both hCdc34- and hRad6B-dependent ubiquitin-mediated proteolysis of hICERIIgamma and hATF5. This degradation requires an active ubiquitin-conjugating enzyme and results in abrogation of ICERIIgamma- and ATF5-mediated repression of cAMP-induced transcription. Consistent with these results, the endogenous ICER protein is elevated in cells which are null for murine Rad6B (mHR6B-/-) or transfected with dominant negative and antisense constructs of human CDC34. Based on the requirement for CREM/ICER and Rad6B proteins in spermatogenesis, we determined expression of Cdc34, Rad6B, CREM/ICER isoforms, and the Skp1-Cullin-F-box ubiquitin protein ligase subunits Cul-1 and Cul-2, which are associated with Cdc34 activity during murine testicular development. Cdc34, Rad6B, and the Cullin proteins are expressed in a developmentally regulated manner, with distinctly different patterns for Cdc34 and the Cullin proteins in germ cells. The Cdc34 and Rad6B proteins are significantly elevated in meiotic and postmeiotic haploid germ cells when chromatin modifications occur. Thus, the stability of specific mammalian transcription factors is the result of complex targeting by multiple ubiquitin-conjugating enzymes and may have an impact on cAMP-inducible gene regulation during both meiotic and mitotic cell cycles.

Presence of salmon gonadotropin-releasing hormone (GnRH) and compounds with GnRH-like activity in the ovary of goldfish.

The presence of ovarian GnRH and/or compounds with GnRH-like activity was investigated in the goldfish ovary. Goldfish ovary was extracted using an acetone/HCl/ether mixture and was purified by Waters C-18 Sep-Pak columns (ovarian extract). The goldfish ovarian extract was found to 1) stimulate gonadotropin release and subunit messenger RNA production in the goldfish pituitary that was inhibited by a GnRH antagonist; 2) stimulate germinal vesicle breakdown in the prophase-I arrested follicle-enclosed goldfish oocytes in vitro, which was inhibited by a GnRH antagonist; 3) immunoreact with various GnRH antisera; and 4) bind to GnRH receptors in the goldfish pituitary and ovary as well as rat pituitary. Further purification with HPLC revealed the presence of two compounds with GnRH-like activity. One with identical chromatographic characteristics, amino acid composition, and primary structure to that of the salmon GnRH (sGnRH), and the other a novel compound with GnRH-like activity.

Characteristics of GnRH binding in the gonads and effects of lamprey GnRH-I and -III on reproduction in the adult sea lamprey.

In the present study, both lamprey GnRH-I and -III stimulated steroidogenesis and induced ovulation in adult female sea lampreys during their final reproductive stage. One injection of lamprey GnRH-III at 0.1 or 0.2 microg/g lamprey stimulated plasma estradiol levels in lampreys held at each of three water temperatures, 13 degrees , 17 degrees , and 19 degrees , corresponding to increasing stages of maturation. Four successive injections, 3 to 4 days apart, of lamprey GnRH-III at 0.1 or 0.2 microg/g body weight induced ovulation in 100 or 88% of lampreys, respectively, compared to 21% in controls by Day 31. Lamprey GnRH-III also had a direct stimulatory effect on estradiol production in the sea lamprey gonads in vitro. Lamprey GnRH-III at 100 or 1000 ng/ml stimulated estradiol levels in media incubated with either lamprey ovaries or testes. In contrast to a previous finding in which lamprey GnRH-III was more potent than lamprey GnRH-I in inducing spermiation in adult male sea lampreys (Deragon and Sower, 1994), the results from the present study indicate that lamprey GnRH-I and -III are equally potent in inducing ovulation and stimulating steroidogenesis in female sea lampreys. In addition, GnRH binding sites have been demonstrated for the first time in both the testis and the ovary of the adult sea lamprey using an analog of mammalian GnRH ([D-Lys6] mammalian GnRH) as a labeled ligand. Scatchard analysis suggested the presence of a high affinity binding site in both the testis and the ovary. In summary, lamprey GnRH-III is biologically active in stimulating the pituitary-gonadal axis in adult female sea lampreys. This is the first report demonstrating the presence of a GnRH binding site in the gonads of an Agnathan. The evidence for a direct stimulatory effect of lamprey GnRH in the gonads, the presence of GnRH binding site, and the absence of GnRH in the plasma suggest that, like other vertebrates including rat, rabbit, teleost fish, and human, there may be a GnRH-like factor produced in the gonads of the lamprey and it may act as a paracrine/autocrine modulator of gonadal function. This study further strengthens the paracrine regulatory role of GnRH peptides in the gonads of vertebrates, which appear to be evolutionarily conserved.

Reconstitution of a MEC1-independent checkpoint in yeast by expression of a novel human fork head cDNA.

A novel human cDNA, CHES1 (checkpoint suppressor 1), has been isolated by suppression of the mec1-1 checkpoint mutation in Saccharomyces cerevisiae. CHES1 suppresses a number of DNA damage-activated checkpoint mutations in S. cerevisiae, including mec1, rad9, rad24, dun1, and rad53. CHES1 suppression of sensitivity to DNA damage is specific for checkpoint-defective strains, in contrast to DNA repair-defective strains. Presence of CHES1 but not a control vector resulted in G2 delay after UV irradiation in checkpoint-defective strains, with kinetics, nuclear morphology, and cycloheximide resistance similar to those of a wild-type strain. CHES1 can also suppress the lethality, UV sensitivity, and G2 checkpoint defect of a mec1 null mutation. In contrast to this activity, CHES1 had no measurable effect on the replication checkpoint as assayed by hydroxyurea sensitivity of a mec1 strain. Sequence analysis demonstrates that CHES1 is a novel member of the fork head/Winged Helix family of transcription factors. Suppression of the checkpoint-defective phenotype requires a 200-amino-acid domain in the carboxy terminus of the protein which is distinct from the DNA binding site. Analysis of CHES1 activity is most consistent with activation of an alternative MEC1-independent checkpoint pathway in budding yeast.

Presence and activity of compounds with GnRH-like activity in the ovary of seabream Sparus aurata.

The binding and activity of gonadotropin-releasing hormone (GnRH) were characterized in the mature gilthead seabream (Sparus aurata) ovary by use of an analogue of salmon GnRH([D-Arg6,Trp7,Leu8,Pro9-N(Et)]GnRH, sGnRH-A) as labeled ligand. The binding of 125I-sGnRH-A to the seabream ovarian membrane preparation was saturable, displaceable, reversible, and dependent on time, temperature and tissue concentration. Addition of unlabeled s-GnRH-A displaced the radio-ligand in a dose-related manner, indicating the presence of one class of high-affinity binding sites with an equilibrium dissociation constant of 45.5 +/- 6.2 nM. Addition of other GnRH peptides, including salmon GnRH ([Trp7,Leu8]GnRH) and chicken GnRH-II ([His5,Trp7,Tyr8]GnRH), also displaced 125I-sGnRH-A; all these peptides bound with lower affinities than sGnRH-A to the seabream ovarian binding site. In this study, we also demonstrated the presence of compounds with GnRH-like activity in the ovary of seabream. Seabream ovarian extract stimulated pituitary gonadotropin release from the goldfish pituitary and displaced 125I-sGnRH-A binding in the seabream ovary. Furthermore, addition of sGnRH-A to cultured seabream oocytes directly stimulated reinitiation of oocyte meiosis, as indicated by germinal vesicle breakdown. Overall, the present study characterizes GnRH-binding sites in the seabream ovary and supports the hypothesis that GnRH or compounds with GnRH-like activity play an autocrine/paracrine role in the regulation of ovarian function in the seabream ovary.

Inhibition of zinc-induced metallothionein mRNA accumulation by gonadotropin-releasing hormone in human hepatocarcinoma cell line HepG2.

Recently we have demonstrated that the human hepatocellular carcinoma-derived cell lines, HepG2 and HuH7, contain gonadotropin-releasing hormone (gonadoliberin) receptors and respond to various molecular forms of gonadoliberin in terms of suppressed proliferation in vitro. This study provides the first demonstration that gonadoliberin inhibits the zinc-induced production of metallothionein mRNA in HepG2 and HuH7 cells. Administration of gonadoliberin agonist (gonadoliberin-A) inhibited the Zn-induced metallothionein mRNA level in a time-related and dose-related manner. The effect of gonadoliberin-A was found to be specific, because concomitant treatment with a gonadoliberin antagonist (gonadoliberin-ANT) blocked gonadoliberin-A inhibition of metallothionein mRNA accumulation. Furthermore, the gonadoliberin-A-induced inhibition of Zn-mediated metallothionein accumulation was found to correlate closely with suppresion of cell proliferation and [3H]thymidine uptake in these cells. It is known that the metal-binding protein metallothionein plays an important role in tumor cell pathobiology and resistance to chemotherapeutic drugs. The present findings may have important implications in the development of an effective chemotherapy for treatment of human liver cancer, in part, by improving the sensitivity of tumor cells through suppression of metallothionein production by gonadoliberin peptides.

Epidermal growth factor receptor binding and biological activity in the ovary of goldfish, Carassius auratus.

The receptor binding and biological activity of epidermal growth factor-urogastrone (EGF) was characterized in the follicle-enclosed goldfish oocyte. The binding of 125I-labeled mouse EGF (mEGF) to goldfish ovarian membrane preparation was peptide specific, saturable, reversible, and dependent on time and tissue concentration. Binding data analysis indicated the presence of a single class of high-affinity binding sites with an estimated equilibrium dissociation constant of 4.4 +/- 1.8 x 10(-10) M. The 125I-mEGF binding was displaced by unlabeled mEGF and by human recombinant transforming growth factor-alpha (hTGF-alpha). Both mEGF and hTGF-alpha were found to stimulate reinitiation of oocyte meiosis, as indicated by germinal vesicle breakdown (GVBD). Treatment with mEGF and hTGF-alpha stimulated GVBD from a basal level of 8.5 to approximately 30% with an estimated half-maximal effective dose for EGF of 5.80 +/- 0.82 + 10(-10) and for hTGF-alpha, 1.9 +/- 1.0 x 10(-10) M. Furthermore, treatment with mEGF marginally increased 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (DHP)-induced GVBD without significantly influencing the gonadotropin-induced response. Treatment with either mEGF or hTGF-alpha significantly reduced human chorionic gonadotropin-stimulated testosterone production in a concentration-related manner. These data suggest that members of the EGF family may play a role in the regulation of ovarian function in goldfish.

Inhibition of human hepatocarcinoma cell proliferation by mammalian and fish gonadotropin-releasing hormones.

This study provides the first demonstration that human hepatocarcinoma-derived cell line HepG2 contains GnRH receptors and responds to various molecular forms of GnRH in terms of inhibition of proliferation in a time- and dose-related manner. In addition, GnRH peptides also significantly inhibited the uptake of [3H]thymidine by these cells. The effect of GnRH was specific because GnRH antagonists reversed the GnRH-mediated inhibition, and non-GnRH peptides had no effect on HepG2 cell proliferation. An important finding is that certain fish GnRH molecules such as lamprey GnRH, which has little gonadotropin (LH) release activity in mammals, suppress hepatocarcinoma cell proliferation with similar potency to a superactive mammalian GnRH analog, [D-Lys6]GnRH. These findings may have profound implications in the development of an effective chemotherapy for treatment of human liver cancer. An added advantage is that fish GnRH forms will likely exert little side effect in terms of human pituitary gonadotropin release, gonadal steroidogenesis, and reproductive functions in general.

Presence of gonadotropin-releasing hormone (GnRH) binding sites and compounds with GnRH-like activity in the ovary of African catfish, Clarias gariepinus.

GnRH binding was characterized in the African catfish ovary by use of an analog of salmon GnRH (sGnRH- ; [D-Arg6, Trp7, Leu8, Pro9-NEt]-GnRH) as a labeled ligand. Binding of sGnRH-A to catfish ovarian membrane preparation was found to be saturable, displaceable, reversible, and dependent on time, temperature, and tissue concentration. Optimal binding was achieved after 70 min of incubation at room temperature (approximately 22 degrees C) at pH 7.6. Addition of unlabeled sGnRH-A displaced the bound 125I-sGnRH-A in a dose-related manner. Hill plot as well as Scatchard analysis indicated the presence of one class of high-affinity binding sites with a equilibrium dissociation constant (Kd) of 0.27 +/- 0.036 nM. Bound 125I-sGnRH-A was also found to be displaceable by catfish GnRH (cfGnRH; [His5, Leu7, Asn8]-GnRH), chicken GnRH-II (cGnRH-II; [His5, Trp7, Tyr8]-GnRH), and salmon GnRH (sGnRH; [Trp7, Leu8]-GnRH); all the peptides were found to bind with lower affinities than sGnRH-A to the catfish ovarian GnRH binding sites. Further experiments using ovarian extracts indicated the presence of compounds with GnRH-like activity in the ovary of African catfish. The crude ovarian extract was found to stimulate pituitary gonadotropin release from goldfish pituitary, as well as displacing 125I-sGnRH-A binding in the catfish ovary. HPLC analysis of the catfish ovarian extract revealed the presence of two fractions that bind specifically to the catfish ovary and release gonadotropin from cultured goldfish pituitary. These fractions include an early eluting peak that does not correspond with the retention time of known GnRH forms in addition to a fraction that co-elutes with the mammalian GnRH. Overall, the study provided characterization of GnRH binding sites in the catfish ovary, and evidence for the presence of compounds with GnRH-like activity in the catfish ovary.

Characterization of gonadotropin-releasing hormone receptors in goldfish ovary: variation during follicular development.

Gonadotropin-releasing hormone (GnRH) receptors were characterized in the goldfish ovary using an analogue of salmon GnRH ([D-Arg6,Trp7,Leu8,Pro9-NEt]GnRH; sGn-RH-A) as a labeled ligand. Binding of sGnRH-A to goldfish follicular membrane preparation was found to be saturable, reversible, and dependent on time, temperature, tissue concentration, and pH. Addition of unlabeled sGnRH-A displaced the bound 125I-labeled sGnRH-A in a dose-related manner. Hill plot as well as Scatchard analysis indicated the presence of two classes of binding sites, a high-affinity/low-capacity site and a low-affinity/high-capacity site, in the fully mature and less mature goldfish ovary. However, immature goldfish ovary contained only a single class of low-affinity binding sites. The equilibrium association constants (Ka) of the low-affinity sites were found to be similar in all follicular groups. However, there was a tendency for a higher Ka value of the high-affinity sites in the less mature follicles (0.5-0.95 mm) compared with the fully mature follicles (1.11-1.48 mm), although the differences were not statistically significant. Bound 125I-sGnRH-A was also found to be displaceable by sGnRH ([Trp7,Leu8]GnRH) and chicken GnRH-II (cGnRH-II; [His5,Trp7,Tyr8]GnRH), which occur naturally in the goldfish brain. sGnRH-A and cGnRH-II were found to bind with greater affinity than sGnRH to the goldfish ovarian GnRH binding sites.