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Supplementary Methods and Figures 1-8 from A Gonadotropin-Releasing Hormone-II Antagonist Induces Autophagy of Prostate Cancer Cells

Gonadotropin-releasing hormone-I (GnRH-I) is known to directly regulate prostate cancer cell proliferation. However, the role of GnRH-II in prostate cancer is unclear. Here, we investigated the effect of the GnRH-II antagonist trptorelix-1 (Trp-1) on growth of PC3 prostate cancer cells. Trp-1 induced growth inhibition of PC3 cells in vitro and inhibited growth of PC3 cells xenografted into nude mice. FITC-N3, an FITC-conjugated Trp-1 analogue, was largely present in the mitochondria of prostate cancer cells, but not in other cells that are not derived from the prostate. Trp-1–induced PC3 growth inhibition was associated with decreased mitochondrial membrane potential and increased levels of mitochondrial and cytosolic reactive oxygen species (ROS). Growth inhibition was partially prevented by cotreating cells with N-acetyl cysteine, an antioxidant. Cytochrome c release and caspase-3 activation were not detected in Trp-1–treated cells. However, Trp-1 induced autophagosome formation, as seen by increased LysoTracker staining and recruitment of microtubule-associated protein 1 light chain 3 to these new lysosomal compartments. Trp-1–induced autophagy was accompanied by decreased AKT phosphorylation and increased c-Jun NH2 terminal kinase phosphorylation, two events known to be linked to autophagy. Taken together, these data suggest that Trp-1 directly induces mitochondrial dysfunction and ROS increase, leading to autophagy of prostate cancer cells. GnRH-II antagonists may hold promise in the treatment of prostate cancer. [Cancer Res 2009;69(3):923–31]

The first total syntheses of isocorniculatolide B, corniculatolide B, and corniculatolide C, consisting of isomeric corniculatolide skeletons, have been accomplished in a divergent manner. The key features of the synthesis involve the construction of diaryl ether linkages by nucleophilic aromatic substitution, installation of a C14-substituted alkyl side chain via a sequence of Baeyer-Villiger reaction and Claisen rearrangement, and efficient construction of corniculatolide and isocorniculatolide frameworks, including 17-membered (exterior) macrolactone skeletons from a versatile diaryl ether intermediate by Mitsunobu macrolactonization. Moreover, we prepared the structural congeners of isomeric corniculatolides via diverted total synthesis approach including desmethyl analogues and related dimeric macrolides. The anti-inflammatory activities of the synthesized natural products, analogues and synthetic intermediates were also investigated. In particular, corniculatolide B significantly inhibited the protein expression of COX-2 and the mRNA expressions of TNF-α, IL-1β and IL-6 by inhibiting of NF-κB signaling in intestinal epithelial cells induced by lipopolysaccharide treatment. It also significantly inhibited the promoter activity and the phosphorylation of subunits p50 and p65 of NF-κB to the same extent as Bay 11-7082, a potent IκB kinase inhibitor. These results suggest that corniculatolide B might have therapeutic potential in inflammatory bowel disease via NF-κB signaling pathway.

Steroidogenic acute regulatory protein (StAR) transfers cholesterol from the outer mitochondrial membrane to the inner membrane where the cytochrome P450 side chain cleavage enzyme (P450scc) resides. This process is the rate-limiting step in steroidogenesis. StAR cDNAs have been cloned and characterized from a range of different species. To investigate the role of StAR in the amphibian system, we cloned a full-length StAR cDNA from bullfrog (Rana catesbeiana) using reverse transcription polymerase chain reaction (RT-PCR) in conjunction with rapid amplification of cDNA ends (RACE). The putative full-length bullfrog StAR (bfStAR) cDNA was 1862 base pairs (bp) in length, and the longest open reading frame (ORF) encoded a protein of 284 amino acids. Amino acid sequence comparison showed that amphibian StAR has a high degree of sequence identity, ranging from 62% to 98%, with StAR proteins of other species. Similar to other species, bfStAR contained two conserved domains, the mitochondrial targeting domain and cholesterol-binding domain, in the N-terminus and C-terminus of the protein, respectively. Northern blot analysis and RT-PCR indicated that StAR mRNA is expressed in the gonads and adrenal gland. Transfection of green monkey kidney (COS-1) cells with an expression construct for bfStAR revealed that it encoded 34 and 27 kDa proteins that were recognized by antiserum raised against the human StAR-related lipid transfer (START) domain.

Neurotensin (NTS) plays important roles in neurotransmission and neuromodulation in the nervous system. NTS exerts its effects mainly by binding to the neurotensin receptor 1 (NTSR1) and receptor 2 (NTSR2) that belong to the G protein-coupled receptor superfamily. While studies on NTS and NTSR have been conducted mainly in mammalian systems, little is known about this ligand-receptor pair in nonmammalian species. Using a basic local alignment search tool combined with our previous identification of bullfrog Lithobates catesbeianus NTSR1 and NTSR2, we can define the evolutionary lineage of NTS and NTSR in vertebrates. Fish may have only one NTSR, which is orthologous to amphibian and mammalian NTSR1. Amphibian and mammalian species have two lineages of NTSR1 and NTSR2 subfamilies. While amphibian and mammalian NTSRs have overall structural similarity within the given subfamilies, they exhibit different pharmacological features and signal transduction pathways. This review will discuss the phylogenetic history of the G protein-coupled NTSRs, the structural features that may influence their pharmacological properties and signal transduction mechanisms, and the molecular interactions between NTSR1 and NTSR2 in vertebrates.

Gonadotropin-releasing hormone-I (GnRH-I) is known to directly regulate prostate cancer cell proliferation. However, the role of GnRH-II in prostate cancer is unclear. Here, we investigated the effect of the GnRH-II antagonist trptorelix-1 (Trp-1) on growth of PC3 prostate cancer cells. Trp-1 induced growth inhibition of PC3 cells in vitro and inhibited growth of PC3 cells xenografted into nude mice. FITC-N3, an FITC-conjugated Trp-1 analogue, was largely present in the mitochondria of prostate cancer cells, but not in other cells that are not derived from the prostate. Trp-1–induced PC3 growth inhibition was associated with decreased mitochondrial membrane potential and increased levels of mitochondrial and cytosolic reactive oxygen species (ROS). Growth inhibition was partially prevented by cotreating cells with N-acetyl cysteine, an antioxidant. Cytochrome c release and caspase-3 activation were not detected in Trp-1–treated cells. However, Trp-1 induced autophagosome formation, as seen by increased LysoTracker staining and recruitment of microtubule-associated protein 1 light chain 3 to these new lysosomal compartments. Trp-1–induced autophagy was accompanied by decreased AKT phosphorylation and increased c-Jun NH2 terminal kinase phosphorylation, two events known to be linked to autophagy. Taken together, these data suggest that Trp-1 directly induces mitochondrial dysfunction and ROS increase, leading to autophagy of prostate cancer cells. GnRH-II antagonists may hold promise in the treatment of prostate cancer. [Cancer Res 2009;69(3):923–31]

A series of small compounds acting at the orphan G protein-coupled receptor GPR92 were screened using a signaling pathway-specific reporter assay system. Lipid-derived molecules including farnesyl pyrophosphate (FPP), N-arachidonylglycine (NAG), and lysophosphatidic acid were found to activate GPR92. FPP and lysophosphatidic acid were able to activate both Gq/11- and Gs-mediated signaling pathways, whereas NAG activated only the Gq/11-mediated signaling pathway. Computer-simulated modeling combined with site-directed mutagenesis of GPR92 indicated that Thr97, Gly98, Phe101, and Arg267 of GPR92 are responsible for the interaction of GPR92 with FPP and NAG. Reverse transcription-PCR analysis revealed that GPR92 mRNA is highly expressed in the dorsal root ganglia (DRG) but faint in other brain regions. Peripheral tissues including, spleen, stomach, small intestine, and kidney also expressed GPR92 mRNA. Immunohistochemical analysis revealed that GPR92 is largely co-localized with TRPV1, a nonspecific cation channel that responds to noxious heat, in mouse and human DRG. FPP and NAG increased intracellular Ca2+ levels in cultured DRG neurons. These results suggest that FPP and NAG play a role in the sensory nervous system through activation of GPR92.

Mesotocin (MT) and vasotocin (VT) are the nonmammalian orthologs of mammalian oxytocin (OT) and arginine vasopressin (AVP), respectively. The OT/AVP family of peptides has arisen from gene duplication but has evolved to possess high selectivity toward their cognate receptors. The process of molecular evolution of receptors to confer high selectivity to their cognate ligands, however, is poorly understood. We constructed a series of reciprocal chimeras using a pair of bullfrog MT receptor (MTR) and VT1 receptor (VT1R) DNA fragments. Among the MTR/VT1R chimeras, the MTR chimera containing a region from transmembrane domain (TMD) VI to the carboxyl-terminal tail (C-tail) of VT1R showed an increased sensitivity to VT, while a chimeric VT1R containing TMD VI to C-tail of MTR showed an increased sensitivity to MT. Further dissection of domains using additional chimeras demonstrated that the receptor with the fragment containing extracellular loop 3 (ECL3) and ECL3-proximal TMDs VI and VII of MTR increased MT selectivity. This fragment is also important for receptor conformation that permits the signaling ability of the receptor. Particularly, the amino acids Val/Ile(6.54) in TMD VI and Pro/Glu(7.29) in ECL3 appear to be involved in this activity, since double mutation of these amino acids completely blocked signaling activity while maintaining ligand binding activity. Mutations at these residues in human OT and AVP 1a receptors markedly decreased receptor signaling activity. This study provides clues for understanding molecular coevolution of the OT/AVP peptides and their receptors with regard to receptor-ligand binding and receptor signaling activity.

The effects of butyltin compounds on follicular steroidogenesis in amphibians were examined using ovarian follicles of Rana catesbeiana . Isolated follicles were cultured for 18 h in the presence and absence of frog pituitary homogenate (FPH) or various steroid precursors, and the steroid levels in the follicles or culture media were measured by radioimmunoassay (RIA). Among the butyltin compounds, tributyltin (TBT) strongly inhibited the FPH-induced synthesis of pregnenolone (P 5 ), progesterone (P 4 ) and testosterone (T). It also inhibited the conversion of P 5 –P 4 and T to estradiol-17β(E 2 ) and it partially suppressed the conversion of androstenedione (AD) to T, but not P 4 to 17α-hydroxyprogesterone (17α-OHP 4 ). A high concentration of dibutyltin (DBT) also inhibited steroidogenesis by the follicles while monobutyltin (MBT) and tetrabutylin (TeBT) had no effect. These results suggest that the initial step of steroidogenesis (P 5 synthesis) and enzymes such as 3β-HSD, 17β-HSD and aromatase are inhibited by TBT or DBT. However, 17α-hydroxylase was not suppressed by TBT or the other butyltin compounds.

Recently, we cloned many of the bullfrog neuropeptide G protein-coupled receptors (GPCRs), including receptors for vasotocin (VT), mesotocin, gonadotropin-releasing hormone (GnRH), neurotensin, apelin, and metastin. Bullfrog GPCRs usually have high affinity for bullfrog ligands but relatively low affinity for mammalian ligands. Reciprocally, synthetic agonists and antagonists developed based upon mammalian ligands display lower affinity at bullfrog receptors. Studies using chimeric or domain-swapped receptors indicate that the motifs responsible for differential ligand selectivity usually reside within transmembrane domain 6 (TMD6)-extracellular loop 3 (ECL3)-transmembrane domain 7 (TMD7). Triple mutation of mammalian V1aR (Phe(6.51) to Tyr, Ile(6.53) to Thr, and Pro(7.33) to Thr) increases VT affinity but greatly reduces arginine vasopressin affinity. This binding profile is similar to that of bullfrog VT1R. Changing just three amino acids in the bullfrog GnRH receptor-1 (i.e. Ser-Gln-Ser in the ECL3) to those found in the type-I mammalian GnRH receptor (i.e. Ser-Glu-Pro) reverses GnRH selectivity. In conclusion, specific receptor motifs that govern ligand selectivity can be determined by comparative molecular analyses of GPCRs and their ligands. Such analysis provides clues for understanding how GPCRs maintain high affinity to their authentic ligands.

Gonadotropin‐releasing hormone (GnRH), synthesized in the hypothalamus, plays a pivotal role in the regulation of vertebrate reproduction. Since molecular isoforms of GnRH and their receptors (GnRHR) have been isolated in a broad range of vertebrate species, GnRH and GnRHR provide an excellent model for understanding the molecular co‐evolution of a peptide ligand‐receptor pair. Vertebrate species possess multiple forms of GnRH, which have been created through evolutionary mechanisms such as gene/chromosome duplication, gene deletion and modification. Similar to GnRHs, GnRH receptors (GnRHR) have also been diversified evolutionarily. Comparative ligand‐receptor interaction studies for non‐mammalian and mammalian GnRHRs combined with mutational mapping studies of GnRHRs have aided the identification of domains or motifs responsible for ligand binding and receptor activation. Here we discuss the molecular basis of GnRH‐GnRHR co‐evolution, particularly the structure‐function relationship regarding li...

Recently, we have shown that some endocrine disruptors, heavy metals, organotins and azoles suppressed steroidogenic enzymes such as P450 side‐chain cleavage enzyme (P450scc) and aromatase in bullfrog ovarian follicles. In the present study, by using an amphibian ovarian follicle culture system, we examined the effects of these endocrine disruptors on maturation and ovulation of oocytes from Rana dybowskii in vitro. Ovarian fragments or isolated follicles were cultured for 24 h in a medium containing frog pituitary homogenate (FPH) or progesterone (P4) with or without endocrine disruptors, and oocyte maturation (germinal vesicle breakdown, GVBD) and ovulation were examined. Among the organotins, tributyltin (TBT) strongly inhibited both FPH‐ and P4‐induced oocyte maturation (ED50: 0.6 and 0.7 μM, respectively); however, tetrabutyltin (TTBT) and dibutyltin (DBT) showed only partial suppression, while monobutyltin (MBT) showed no inhibitory effect. All of the organotins suppressed P4‐induced oocyte...

The neurohypophysial nonapeptides vasopressin (VP) and oxytocin (OT) modulate a broad range of cognitive and social activities. Notably, in amphibians, vasotocin (VT), the ortholog of mammalian VP, plays a crucial role in the control of sexual behaviors. Because several neurosteroids also regulate reproduction-related behaviors, we investigated the possible effect of VT and the OT ortholog mesotocin (MT) in the control of neurosteroid production. Double immunohistochemical labeling of frog brain sections revealed the presence of VT/MT-positive fibers in close proximity of neurons expressing the steroidogenic enzymes 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase (3beta-HSD) and cytochrome P450 17alpha-hydroxylase/c17, 20-lyase (P450(C17)). High concentrations of VT and MT receptor mRNAs were observed in diencephalic nuclei containing the 3beta-HSD and P450(C17) neuronal populations. Exposure of frog hypothalamic explants to graded concentrations of VT or MT produced a dose-dependent increase in the formation of progesterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, and dehydroepiandrosterone. The stimulatory effect of VT and MT on neurosteroid biosynthesis was mimicked by VP and OT, as well as by a selective V1b receptor agonist, whereas V2 and OT receptor agonists had no effect. VT-induced neurosteroid production was completely suppressed by selective V1a receptor antagonists and was not affected by V2 and OT receptor antagonists. Concurrently, the effect of MT on neurosteroidogenesis was markedly attenuated by selective OT and V1a receptor antagonists but not by a V2 antagonist. The present study provides the first evidence for a regulatory effect of VT and MT on neurosteroid biosynthesis. These data suggest that neurosteroids may mediate some of the behavioral actions of VT and MT.

The basic leucine zipper transcription factor, CCAAT enhancer-binding protein-alpha (C/EBPalpha), negatively regulates cell proliferation and induces terminal differentiation of various cell types. C/EBPalpha is expressed in the prostate, but its potential role in the tissue is unknown. Herein, we show that C/EBPalpha is highly expressed at the stage of growth arrest during prostate development. Furthermore, overexpression of C/EBPalpha decreases the rate of DNA synthesis in LNCaP prostate cancer cells. Investigation of the potential cross-talk between C/EBPalpha and androgen receptor (AR) that is responsible for androgen-dependent prostate proliferation demonstrates that androgen-dependent transactivation of AR is strongly repressed by C/EBPalpha. C/EBPalpha directly binds AR in vitro and forms a complex with AR in vivo. C/EBPalpha neither prevents the nuclear translocation of AR nor disrupts the N/C-terminal interaction of AR, which are both necessary for its proper transactivation activity upon ligand binding. To modulate AR transactivation, however, C/EBPalpha does compete with AR coactivators for AR binding. Additionally, C/EBPalpha is recruited onto AR-target promoters with AR and is further able to inhibit the expression of endogenous prostate-specific antigen in prostate cancer cells. Our results suggest C/EBPalpha as a potent AR corepressor and provide insight into the role of C/EBPalpha in prostate development and cancer.

Azoles are widely used antifungal agents, which inhibit the biosynthesis of fungal cell‐membrane ergosterol. In this study, using an amphibian follicle culture system, the effects of azoles on follicular steroidogenesis in frogs were examined. Itraconazole (ICZ), clotrimazole (CTZ) and ketoconazole (KCZ) suppressed pregnenolone (P5) production by the follicles (ED50; 0.04 μM, 0.33 μM, and 0.91 μM, respectively) in response to frog pituitary homogenates (FPH). However, fluconazole (FCZ), miconazole (MCZ) and econazole (ECZ) were not effective in the suppression of P5 production. Not all the azoles examined suppressed the conversion of exogenous P5 to progesterone (P4) (by 3β‐HSD) or P4 to 17α‐hydroxyprogesterone (17α‐OHP) (by 17α‐hydroxylase), or androstenedione (AD) to testosterone (T) (by 17β‐HSD). In contrast, CTZ, MCZ and ECZ in medium partially suppressed the conversion of 17α‐OHP to AD (by C17‐20 lyase) (ED50; 0.25 μM, 4.5 μM, and 0.7 μM, respectively) and CTZ, KCZ, ECZ and MCZ strongly supp...

Context: GnRH is known to directly regulate prostate cancer cell proliferation, but the precise mechanism of action of the peptide is still under investigation.Objective: This study demonstrates differential effects of GnRH-I and GnRH-II on androgen-independent human prostate cancer cells.Results: Both GnRH-I and GnRH-II increased the intracellular Ca2+ concentration ([Ca2+]i) either through Ca2+ influx from external Ca2+ source or via mobilization of Ca2+ from internal Ca2+ stores. Interestingly, the [Ca2+]i increase was mediated by activation of the ryanodine receptor but not the inositol trisphosphate receptor. Trptorelix-1, a novel GnRH-II antagonist but not cetrorelix, a classical GnRH-I antagonist, completely inhibited the GnRH-II-induced [Ca2+]i increase. Concurrently at high concentrations, trptorelix-1 and cetrorelix inhibited GnRH-I-induced [Ca2+]i increase, whereas at low concentrations they exerted an agonistic action, inducing Ca2+ influx. High concentrations of trptorelix-1 but not cetrorelix-induced prostate cancer cell death, probably through an apoptotic process. Using photoaffinity labeling with 125I-[azidobenzoyl-d-Lys6]GnRH-II, we observed that an 80-kDa protein specifically bound to GnRH-II.Conclusions: This study suggests the existence of a novel GnRH-II binding protein, in addition to a conventional GnRH-I receptor, in prostate cancer cells. These data may facilitate the development of innovatory therapeutic drugs for the treatment of prostate cancer.

The SWI3-related gene product (SRG3), a component of the mouse SWI/SNF complex, has been suggested to have an alternative function. Here, we demonstrate that in the prostate transactivation of the androgen receptor (AR) is modulated by SRG3 in multiple ways. The expression of SRG3, which is developmentally regulated in the prostate, is induced by androgen through AR. SRG3 in turn enhances the transactivation of AR, providing a positive feedback regulatory loop. The SRG3 coactivation of AR transactivation is achieved through the recruitment of coactivator SRC-1, the protein level of which is upregulated by SRG3, providing another pathway of positive regulation. Interestingly, SRG3 coactivation of AR transactivation is fully functional in BRG1/BRM-deficient C33A cells and the AR/SRG3/SRC-1 complex formed in vivo contains neither BRG1 nor BRM protein, suggesting the possibility of an SRG3 function independent of the SWI/SNF complex. Importantly, the AR/SRG3/SRC-1 complex occupies androgen response elements on the endogenous SRG3 and PSA promoter in an androgen-dependent manner in mouse prostate and LNCaP cells, respectively, inducing gene expression. These results suggest that the multiple positive regulatory mechanisms of AR transactivation by SRG3 may be important for the rapid proliferation of prostate cells during prostate development and regeneration.

Recently, we demonstrated that the mammalian type-I GnRH receptor (GnRHR) has a high preference for the phospholipase C/protein kinase C (PLC/PKC)-linked signaling pathway, whereas non-mammalian bullfrog (bf) GnRHRs couple to both adenylate cyclase/protein kinase A (AC/PKA)- and PLC/PKC-linked signaling pathways. In the pre-sent study, using AC/PKA-specific reporter (cAMP-responsive element-luciferase) and PLC/PKC-specific reporter (serum-responsive element-luciferase) systems, we attempted to identify the motif responsible for this difference. A deletion of the intracellular carboxyl-terminal tail (C tail) of bfGnRHR-1 remarkably decreased its ability to induce the AC/PKA-linked signaling pathway. Further dissection of the C tail indicated that an HFRK motif in the membrane-proximal sequence of bfGnRHR-1 C tail is a minimal requirement for the AC/PKA-linked signaling pathway as the addition of this motif to rat GnRHR or deletion of it from bfGnRHR-1 significantly affected the ability to induce the AC/PKA-linked signaling pathway. Deletion or addition of the HFRK motif, however, did not critically influence the PLC/PKC-linked signaling pathway. These results indicate that the HFRK motif in the membrane-proximal region confers the differential signal transduction pathways between mammalian and nonmammalian GnRHRs.

Mammalian spermatogenesis is orchestrated by many specific molecular and cellular events. To understand the detailed mechanism by which spermatogenesis is controlled, the specific genes involved in this process must be identified and studied. From the subtracted cDNA library of rat testis prepared using the representational difference analysis (RDA) method, we isolated the cDNA clone of steroidogenic acute regulatory (StAR) protein-related lipid transfer (START) protein 6 (Stard6). Stard6 cDNA consists of 1146 base pairs of nucleotides and has the longest open reading frame, of 227 amino acids. Northern blot analysis revealed Stard6 mRNA to be testis-specific. The mRNA transcript appeared from the third week of postnatal development, and the expression level increased up to adulthood. Moreover, in situ hybridization showed Stard6 mRNA expression to be germ cell-specific and expressed only during the maturation stages of round and elongated spermatids of adult rat testis. Western blot analysis with Stard6 antibody revealed a 28-kDa Stard6 protein only in testis. Immunohistochemistry further confirmed localization of Stard6 protein expressed in mature germ cells, in concert with the in situ hybridization result. Taken together, these results suggest that Stard6, a member of the START protein family, may play a role during germ cell maturation in adult rat testis.

Mammalian type I and II gonadotropin-releasing hormone (GnRH) receptors (GnRHRs) show differential ligand preference for GnRH-I and GnRH-II, respectively. Using a variety of chimeric receptors based on green monkey GnRHR-2 (gmGnRHR-2), a representative type II GnRHR, and rat GnRHR, a representative type I GnRHR, this study elucidated specific domains responsible for this ligand selectivity. A chimeric gmGnRHR-2 with the extracellular loop 3 (EL3) and EL3-proximal transmembrane helix 7 (TMH7) of rat GnRHR showed a great increase in ligand sensitivity to GnRH-I but not to GnRH-II. Point-mutation studies indicate that four amino acids, Leu/Phe(7.38), Leu/Phe(7.43), Ala/Pro(7.46), and Pro/Cys(7.47) in TMH7 are critical for ligand selectivity as well as receptor conformation. Furthermore, a combinatory mutation (Pro(7.31)-Pro(7.32)-Ser(7.33) motif to Ser-Glu-Pro in EL3 and Leu(7.38), Leu(7.43), Ala(7.46), and Pro(7.47) to those of rat GnRHR) in gmGnRH-2 exhibited an approximately 500-fold increased sensitivity to GnRH-I, indicating that these residues are critical for discriminating GnRH-II from GnRH-I. [Trp(7)]GnRH-I and [Trp(8)]GnRH-I but not [His(5)]GnRH-I exhibit a higher potency in activating wild-type gmGnRHR-2 than native GnRH-I, indicating that amino acids at positions 7 and 8 of GnRHs are more important than position 5 for differential recognition by type I and type II GnRHRs. As a whole, these data suggest a molecular coevolution of ligands and their receptors and facilitate the understanding of the molecular interaction between GnRHs and their cognate receptors.

Arginine vasotocin (VT) is the ortholog in all nonmammalian vertebrates of arginine vasopressin (AVP) in mammals. We have previously cloned an amphibian V1atype vasotocin receptor (VT1R) that exhibited higher sensitivity for VT than AVP, while the mammalian V1a type receptor (V1aR) responded better to AVP than VT. In the present study, we identified the amino acid residues that confer differential ligand selectivity for AVP and VT between rat V1aR and bullfrog VT1R (bfVT1R). A chimeric rat V1aR having transmembrane domain (TMD) VI to the carboxyl-terminal tail (C-tail) of bfVT1R showed a reverse ligand preference for AVP and VT, whereas a chimeric VT1R with TMD VI to the C-tail of rat V1aR showed a great increase in sensitivity for AVP. A single mutation (Ile(315(6.53)) to Thr) in TMD VI of V1aR increased the sensitivity for VT, while a single mutation (Phe(313(6.51)) to Tyr or Pro(334(7.33)) to Thr) reduced sensitivity toward AVP. Interestingly the triple mutation (Phe(313(6.51)) to Tyr, Ile(6.53) to Thr, and Pro(7.33) to Thr) of V1aR increased sensitivity to VT but greatly reduced sensitivity to AVP, behaving like bfVT1R. Further, like V1aR, a double mutant (Tyr(306(6.51)) to Phe and Thr(327(7.33)) to Pro) of bfVT1R showed an increased sensitivity to AVP. These results suggest that Phe/Tyr(6.51), Ile/Thr(6.53), and Pro/Thr(7.33) are responsible for the differential ligand selectivity between rat V1aR and bfVT1R. This information regarding the molecular interaction of VT/AVP with their receptors may have important implications for the development of novel AVP analogs.

The neurohypophysial nonapeptides vasotocin (VT) and mesotocin (MT) are the amphibian counterparts of arginine vasopressin (AVP) and oxytocin (OT). We have here reported the cloning and functional characterization of the receptors for vasotocin (VTR) and mesotocin (MTR) in two species of frog, Rana catesbeiana and Rana esculenta. The frog VTR and MTR cDNAs encode proteins of 419 and 384 amino acids respectively. Frog VTR exhibits a high degree of sequence identity with the mammalian AVP-1a (V1a) receptor while the frog MTR possesses a high degree of sequence identity with the mammalian OT receptor. Activation of VTR induced both c-fos promoter- and cAMP-responsive element (CRE)-driven transcriptional activities, while activation of MTR induced c-fos promoter-driven transcriptional activity but failed to evoke CRE-driven transcriptional activity, suggesting differential G protein coupling between VTR and MTR. The VTR exhibited the highest sensitivity for VT followed by OT>AVP approximately MT, whereas the MTR showed preferential ligand sensitivity for MT>OT>VT>AVP. A V1a agonist but not V2 and OT agonists substantially activated both VTR and MTR with a similar sensitivity. V1a, V2 and OT antagonists inhibited MT-induced MTR activation but not VT-induced VTR activation. In the frog brain, VTR and MTR mRNAs were found to be widely expressed in the telencephalon, diencephalon and mesencephalon, and exhibited very similar regional distribution. In the pituitary, VTR and MTR were expressed in the distal and intermediate lobes but were virtually absent in the neural lobe. Taken together, these data indicated that, although the distribution of VTR and MTR largely overlaps in the frog brain and pituitary, VT and MT may play distinct activities owing to the ligand selectivity and different signaling pathways activated by their receptors.

A Glu/Asp7.32 residue in the extracellular loop 3 of the mammalian GnRH receptor (GnRHR) is known to interact with Arg8 of mammalian GnRH (mGnRH), which may confer preferential ligand selectivity for mGnRH than for chicken GnRH-II (cGnRH-II). However, some nonmammalian GnRHRs also have the Glu/Asp residue at the same position, yet respond better to cGnRH-II than mGnRH. Amino acids flanking Glu/Asp7.32 are differentially arranged such that mammalian and nonmammalian GnRHRs have an S-E/D-P motif and P-X-S/Y motif, respectively. We presumed the position of Ser7.31 or Pro7.33 of rat GnRHR as a potential determinant for ligand selectivity. Either placing Pro before Glu7.32 or placing Ser after Glu7.32 significantly decreased the sensitivity and/or efficacy for mGnRH, but slightly increased that for cGnRH-II in several mutant receptors. Among them, those with a PEV, PES, or SES motif exhibited a marked decrease in sensitivity for mGnRH such that cGnRH-II had a higher potency than mGnRH, showing a reversed preferential ligand selectivity. Chimeric mGnRHs in which positions 5, 7, and/or 8 were replaced by those of cGnRH-II revealed a greater ability to activate these mutant receptors than mGnRH, whereas they were less potent to activate wild-type rat GnRHR than mGnRH. Interestingly, a mutant bullfrog type I receptor with the SEP motif exhibited an increased sensitivity for mGnRH but a decreased sensitivity for cGnRH-II. These results indicate that the position of Pro and Ser near Glu7.32 in the extracellular loop 3 is critical for the differential ligand selectivity between mammalian and nonmammalian GnRHRs.

Progesterone plays a pivotal role in the regulation of reproduction in all vertebrates and binds to nuclear hormone receptor, one of ligand-dependent transcription factors. Although avian and mammalian progesterone receptors (PR) have been well characterized, detail structure and function of amphibian progesterone receptor in wild frog is poorly studied yet. Here we report the cloning and characterization of a novel progesterone receptor from the Korean frog, Rana dybowskii. The R. dybowskii progesterone receptor (dyPR, GenBank Accession No. AF431813) cDNA isolated from testis encodes a protein of 711 amino acids which shows approximately 60% overall identity with the Xenopus progesterone receptor. Reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrates that dyPR is expressed in all the tissues examined. Electrophoretic mobility shift assays demonstrate that this receptor specifically binds to a progesterone response element (PRE), and transient transfection studies demonstrate that dyPR significantly activates the transcription of a PRE containing reporter element. Finally, confocal microscopy demonstrates the localization of this protein in nucleus, cytoplasm, and plasma membrane in transiently transfected CV-1 cell. These results indicate that dyPR cDNA encodes a classical progesterone receptor and molecular characterization of dyPR may provide us new information about the evolution of steroid hormone receptor.

Gonadotropin-releasing hormone (GnRH) regulates the reproductive system through the cognate GnRH receptor (GnRHR) in vertebrates. In this study, we cloned a cDNA encoding the full-length open reading frame sequence for green monkey type-II GnRHR (gmGnRHR-2) from the genomic DNA of CV-1 cells. Transient transfection study showed that gmGnRHR-2 was able to induce both c-fos promoterand cAMP responsive element-driven transcriptional activities, indicating that gmGnRHR-2 couples to both Gs- and Gq/11-linked signaling pathways. gmGnRHR-2 responded better to GnRH-2 ([His 5 ,T rp 7 ,T yr 8 ]GnRH) than GnRH-1 ([Tyr 5 , Leu 7 ,A rg 8 ]GnRH). Substitutions of His 5 ,T rp 7 , and/or Tyr 8 in GnRH-1 increased the potency to activate gmGnRHR-2, suggesting that individual His 5 ,T rp 7 , and Tyr 8 in GnRH-2 contributed to differential ligand sensitivity of gmGnRHR-2. Substitution of d-Ala for Gly 6 in GnRH-2 increased the potency to activate the receptor, suggesting that GnRH-2 has a constrained conformation when it binds to the receptor. GnRH-induced gmGnRHR-2 activation was specifically inhibited by GnRH-2 antagonists, Trptorelix-1 and -2, but not by a GnRH-1 antagonist, Cetrorelix. In conclusion, gmGnRHR-2 revealed preferential ligand selectivity for GnRH-2 and its analogs, suggesting that gmGnRHR-2 has a functional activity that is different from mammalian type-I GnRHRs but similar to non-mammalian GnRHRs. © 2003 Elsevier Ireland Ltd. All rights reserved.

Recently, we have identified three distinct types of gonadotropin-releasing hormone receptor (GnRHR) in the bullfrog (designated bfGnRHR-1, bfGnRHR-2, and bfGnRHR-3). In the present study, we compared G protein coupling preference of mammalian and nonmammalian GnRHRs. In a transient expression system, stimulation of either bfGnRHRs or rat GnRHR by GnRH significantly increased both inositol phosphates (IP) and cAMP productions, but ratios of IP to cAMP induction levels were quite different among the receptors, indicating differential G protein coupling preference. Using cAMP-dependent protein kinase A (PKA)-specific (CRE-luc) or protein kinase C (PKC)-specific reporter (c-fos-luc) systems, we further examined G(s) and G(q/11) coupling preference of these GnRHRs. Since activities of CRE-luc and c-fos-luc were highly dependent on cell types, GnRH-induced CRE-luc or c-fos-luc activity was normalized by forskolin-induced CRE-luc or 12-O-tetradecanoylphenol-13-acetate (TPA)-induced c-fos-luc activity, respectively. This normalized result indicated that bfGnRHR-2 couples to G(s) more actively than G(q/11), while bfGnRHR-1 and -3 couple to G(s) and G(q/11) with similar strength. However, the rat GnRHR appeared to couple to G(q/11) more efficiently than G(s). This study was further confirmed by an experiment in which GnRH augmented CRE-driven luciferase activity in alphaT3-1 cells when CRE-luc was cotransfected with bfGnRHRs but not with vehicle or rat GnRHR. Collectively, these results indicate that mammalian and nonmammalian GnRHRs may induce diverse cellular and physiological responses through differential activation of PKA and PKC signaling pathways.

Recently, we have identified three distinct types of bullfrog GnRH receptor (designated bfGnRHR-1, bfGnRHR-2, and bfGnRHR-3). In the present study, we have isolated three GnRHR clones in Rana dybowskii (dyGnRHR-1, dyGnRHR-2, and dyGnRHR-3). Despite high homology of dyGnRHRs with the corresponding bfGnRHRs, dyGnRHRs revealed different signaling pathways and ligand sensitivity compared with the bfGnRHR counterparts. Activation of dyGnRHRs with GnRH stimulated cAMP-mediated gene expression. However, dyGnRHR-3 but not dyGnRHR-1 and -2 induced c-fos promoter-driven gene expression. Consistently, dyGnRHR-1 and dyGnRHR-2 were not able to increase GnRH-induced inositol phosphate accumulation, whereas all bfGnRHRs and dyGnRHR-3 were, indicating that dyGnRHR-1 and dyGnRHR-2 are coupled to solely Gs, whereas all bfGnRHRs and dyGnRHR-3 are coupled to both Gs and Gq/11. Moreover, dyGnRHR-1 and dyGnRHR-2 showed about 10-fold less sensitivity to each ligand than that of the bfGnRHR counterparts. Using type 1 chimeric and point-mutated receptors, we further elucidated that specific amino acids, Ala/Thr201 in extracellular loop 2 and Leu/Phe290 in transmembrane domain 6 of the type 1 receptor, are responsible for ligand sensitivity and signal transduction pathway. Particularly, substitution of Leu290 to Phe in dyGnRHR-1 increased GnRH-induced inositol phosphate production as well as c-fos promoter-driven gene expression whereas substitution of Phe290 to Leu in bfGnRHR-1 decreased those activities. Collectively, these results demonstrate the presence of three types of GnRHR in amphibians, and suggest species- and type-specific ligand recognition and different signaling pathways in frog GnRHRs.

Recently we characterized three distinct GnRH receptors in the bullfrog (bfGnRHR-1, bfGnRHR-2, and bfGnRHR-3). In the present study, we further investigated the expression and function of splice variants, generated from the primary bfGnRHR-3 transcript by exon skipping (splice variant 1), intron retention (splice variants 2 and 3), and/or transcriptional slippage (splice variant 4), apart from the constitutively spliced form (wild-type). Cellular expression and function of the splice variants were examined using a transient expression system. Immunoblot analysis revealed that the wild-type receptor and all splice variant proteins were expressed in transfected HeLa cells with no significant differences in expression levels. These splice variants showed a very low binding affinity to ligand and did not induce signal transduction in response to GnRH treatment. Interestingly, cotransfection of the wild-type with splice variants 2--4, but not with splice variant 1, significantly inhibited wild-type receptor-mediated signaling. Subcellular localization analysis of green fluorescent protein-tagged wild-type and splice variant proteins revealed that the wild-type receptor protein was mainly localized in the cell membrane, whereas the splice variant 1 protein was exclusively detected in the cytoplasm. The splice variant 2--4 proteins, however, were found in both the cell membrane and cytoplasm. The inhibition of wild-type receptor signaling by splice variants 2--4 and the subcellular localization of splice variants 2-4 suggest a possible physical interaction of splice variants 2--4 with the wild-type receptor protein. In addition, the ratio of mRNA levels of the wild-type to splice variants 2--4 significantly varied from hibernation (wild-typesplice variants 2--4) to the prebreeding season (wild-typesplice variants 2--4). Collectively, these results suggest that alternative splicing of the bfGnRHR-3 primary transcript plays a role in fine-tuning GnRH receptor function in amphibians.

We have previously shown that oocyte maturation is induced by an immobilized progesterone, progesterone‐3‐carboxymethyloxime ‐ bovine serum albumin conjugate (P‐BSA) in Rana dybowskii. In this study, we confirmed the maturation inducing activity of P‐BSA on Xenopus oocyte and examined the binding character of the immobilized progesterone on the surface of Xenopus oocytes after removal of the vitelline layer. P‐BSA induced maturation of Xenopus oocytes but E‐BSA failed to do so as observed in Rana. Binding of the immobilized progesterone, fluorescein isothiocyanate‐labeled progesterone‐3‐O‐carboxymethyloxime‐BSA (P‐BSA‐FITC) on the devitellined oocytes surface was examined by fluorescence confocal microscopy. The binding affinity of P‐BSA‐FITC to the devitellined oocyte was higher than that of estrogen‐BSA‐FITC (E‐BSA‐FITQ or testo‐sterone‐BSA‐FITC (T‐BSA‐FITC). The binding disappeared in the presence of excess free progesterone but not in the presence of free estrogen. Maximum binding occurred after two‐h...

We have isolated the cDNAs encoding the GnRH1 and GnRH2 precursors, respectively, from bullfrog (Rana catesbeiana) brain. The first cDNA consists of 648 bp and contains an open-reading frame of 270 nucleotides, encoding the bullfrog GnRH1 precursor. The second cDNA consists of 1053 bp and contains an open-reading frame of 255 nucleotides, encoding the bullfrog GnRH2 precursor. Both types of bullfrog GnRH precursor have a similar molecular architecture as observed in other GnRH precursors, consisting of a signal peptide, followed by the GnRH decapeptide, a conserved carboxy-terminal amidation and proteolytical processing site, and a GnRH-associated peptide (GAP). In addition, we have identified a third cDNA, containing 24 additional nucleotides in its GAP-coding region. Genomic PCR and sequence analysis confirmed that this cDNA represents an alternative splice variant of the bullfrog GnRH2-precursor pre-mRNA. The bullfrog GnRH1 precursor exhibits 60% and less than 40% amino acid identity to its Xenopus and mammalian counterparts, respectively, whereas the bullfrog GnRH2 precursor displays 50% to 60% amino acid identity to that of its nonmammalian counterparts, but shares only 25% amino acid identity with its mammalian counterparts. Northern blot analysis revealed a single GnRH1-precursor mRNA species of ∼0.75 kilobases, expressed in bullfrog forebrain, and a single GnRH2-precursor mRNA species of ∼1.1 kilobases, expressed in bullfrog midbrain/hindbrain. Furthermore, both bullfrog GnRH-precursor mRNAs exhibited a differential spatiotemporal expression pattern. Genomic Southern blot analysis indicated that both bullfrog GnRH genes are present as single copy genes. This is the first report on the molecular cloning of a GnRH2-precursor cDNA from an amphibian species. In addition, we present data showing that alternative splicing is utilized to generate different GnRH2-precursor mRNAs. J. Exp. Zool. 289:190–201, 2001. © 2001 Wiley-Liss, Inc.

Rana ovarian follicles consist of oocyte, vitelline envelope, granulosa cells, and theca/epithelial layer. Using scanning electron microscopy, the surface structure of each follicular component was investigated. Changes in oocyte surface during oocyte maturation were also examined. Theca/epithelial layer was almost transparent and some blood vessels and granulosa cells were observed underneath in intact follicle. The number of granulosa cells was estimated to be 6700-7200 per oocyte. The granulosa cells partially overlapped each other and their microvilli penetrated the vitelline membrane via holes present in the vitelline envelope and seemed to be linked to oocyte microvilli. After removal of the vitelline envelope by microforcep, oocyte microvilli were observed on the surface of the devitellined oocyte. The oocyte microvilli formed partial clusters on the surface of white spot area which appears iust before germinal vesicle breakdown (GVBD), whereas they were evenly distributed in other areas. The microvilli became shorter and less dense with oocyte maturation. The lengths of oocyte microvilli in the immature and mature oocyte were 1.5 m and 0.6 m, respectively. The present study suggests a fundamental structural change occurring on the oocyte surface during maturation.

To date nine structural variants of GnRH have been identified in vertebrates and two additional forms have been isolated from a tunicate. In amphibians only mammalian GnRH ([Arg 8 ] GnRH) and type II GnRH (chicken GnRH II, [His 5 , Trp 7 , Tyr 8 ] GnRH) have been identified. In the present study, a full-length cDNA encoding a novel type of GnRH was isolated from pituitary of Rana dybowskii. The GnRH gene encodes a GnRH peptide ([Trp 8 ] GnRH) in which tryptophan is substituted for arginine of mammalian GnRH Northern blot analysis revealed the presence of a single 500 bp transcript for the [Trp 8 ] GnRH precursor in forebrain but its absence in testis, ovary, kidney and liver. Restriction digests of genomic DNA demonstrated a single copy of the gene. The [Trp 8 ] GnRH immunoreactive cells were identified in the preoptic area of the frog brain. Synthetic [Trp 8 ] GnRH was tested for its ability to stimulate inositol phosphate production by COS-1 cells transfected with the cloned Xenopus pituitary GnRH receptor and the cloned human GnRH receptor. [Trp 8 ] GnRH had a potency of about 60% compared with mammalian GnRH ([Arg 8 ] GnRH) for the Xenopus receptor, whereas the potency of [Trp 8 ] GnRH was ≈5% compared with mammalian GnRH for the human receptor. Both mammalian GnRH and [Trp 8 ] GnRH were 1000-fold less potent than type II GnRH for the Xenopus GnRH receptor. The similar potency of [Arg 8 ] GnRH and the novel [Trp 8 ] GnRH for the Xenopus pituitary receptor indicates that, unlike the human receptor, the Xenopus receptor does not discriminate between these amino acids in position eight thereby allowing substitution of the arginine in the mammalian GnRH.

Pituitary adenylate cyclase-activating polypeptide (PACAP) regulates pituitary hormone biosynthesis and secretion through its cognate receptors. PACAP also plays an important role in the regulation of ovarian steroid biosynthesis. If so, there might be a feedback regulation of hypothalamic PACAP synthesis by the pituitary and by ovarian steroids. In the present study, we used RNase protection assays to determine changes in mRNA levels of PACAP and type I PACAP receptor (PAC1) under the conditions of ovariectomy and replacement with ovarian steroids. Progesterone (P) alone or in combination with estradiol (E) induced significant increases in PACAP mRNA level in the medial basal hypothalamus (MBH) and PAC1 mRNA levels in MBH and the preoptic area (POA). This finding suggests that feedback regulation takes place between the ovary and hypothalamic PACAP neurons. P is known to be a major regulatory feedback factor for hypothalamic luteinizing hormone-releasing hormone (LHRH) neurons, but P receptor is not present in these neurons. Therefore, we examined a possible involvement of PACAP in the feedback regulatory pathway of P to LHRH neurons. After an antisense PAC1 oligodeoxynucleotide (ODN) was i.c.v.-injected into the third ventricle of E and P-treated rats, LHRH mRNA levels were determined. The ODN markedly decreased the P-induced increase in the LHRH mRNA level. Taken together, the present data suggest that PACAP may play a role as a mediator in the regulation of LHRH synthetic machinery by stimulatory feedback of P.

Pituitary adenylate cyclase activating polypeptide (PACAP) was isolated from ovine hypothalamus and known to stimulate the production of cAMP in anterior pituitary cells. In the recent report, the expression of PACAP was detected in preovulatory follicles, and treatment with PACAP stimulated the production of progesterone and prostaglandin E2 through the action of AC and PLC pathways in the ovary. PACAP binds to three type receptors. Type I A receptor is coupled to adenylate cyclase (AC) and phospholipase C (PLC) pathways, while type I B and type II receptors are only coupled to AC. Thus, the present study aimed to evaluate the temporal expression of PACAP and its type I A receptor mRNAs in the rat ovary after treatment with pregnant mare's serum gonadotropin and human chorionic gonadotropin (hCG). Northern blot analysis showed that PACAP transcripts were transiently expressed from 3–9 hr after hCG treatment, reaching a maximum at 6 hr. During these time points, PACAP mRNAs were specifically and strongly expressed in granulosa cells and cumulus cells of large preovulatory follicles and interstitial glandular cells. Type I A receptor mRNAs were also transiently expressed in granulosa cells of large preovulatory follicles from 3–9 hr after hCG treatment. PACAP and its type I A receptor mRNAs were expressed in the same preovulatory follicles. These results demonstrate that PACAP acts as an autoregulator or pararegulator through type I A receptor in granulosa cells and cumulus cells of large preovulatory follicles. Thus, we suggest that PACAP may have a critical role in granulosa cells of preovulatory follicles for the preparation of ovulation. Mol. Reprod. Dev. 55:379–386, 2000. © 2000 Wiley-Liss, Inc.

In order to understand the mechanism of oocyte maturation in seasonal-breeding wild frogs, we have cloned and sequenced a cDNA encoding Cdc2 kinase, a component of the maturation-promoting factor (MPF) in Rana dybowskii. About 1.2-kb cDNA was isolated by reverse transcription coupled to polymerase chain reaction (RT-PCR) and cDNA library screening. The cloned Rana Cdc2 cDNA encodes a complete open-reading frame with 302 amino acid residues, which deduce a 34-kDa protein. Homology of more than 80% was found between the deduced amino acid sequence of Rana Cdc2 and that of five phylogenetically distant organisms, and 94% identity was found between Rana and Xenopus. More importantly, the Thr14, Tyr15, and Thr161 residues, the phosphorylation sites for the activation of the enzyme, are highly conserved. In vitro -translated Rana Cdc2 cross-reacted with Xenopus p34 cdc2 antibody as shown by Western blot. Northern blot analysis showed that a 1.7-kb transcript was highly expressed in the gonads compared to other tissues, indicating the important role of Cdc2 kinase in gonads as a component of MPF. The cloned Rana Cdc2 cDNA also exhibited histone H1 kinase activity when expressed in CV-1 cells. In the present study, therefore, we have characterized the Rana Cdc2 kinase in amphibian, which will be helpful in understanding the process of oocyte maturation related to the reproduction cycle of wild frogs.

Expression of pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide with considerable homology to vasoactive intestinal peptide, has been shown to be stimulated by gonadotropins in the ovary. The present studies further evaluated the cell-type specific expression and gonadotropin regulation of PACAP type I receptor (PACAPR) messenger RNA in immature rat ovaries and in cultured preovulatory follicles. Northern blot analysis of ovaries obtained from prepubertal rats revealed the increased expression of PACAPR during prepubertal development. The major cell types expressing PACAPR messenger RNA were granulosa cells of large preantral follicles. Treatment of immature rats with PMSG caused a decrease in ovarian PACAPR expression. In contrast, treatment with human (h) CG at 2 days after PMSG treatment stimulated ovarian PACAPR messenger RNA within 3–6 h in granulosa cells of preovulatory follicles. Treatment of cultured preovulatory follicles in vitro with LH further confirmed the time- and ...

Previously, based on studies conducted using Rana nigromaculata, a two-cell model involving the theca and granulosa cells was proposed to account for the steroidogenic activity of amphibian ovarian follicles. Experiments were carried out to ascertain whether the model was applicable to four other frog species with different reproductive cycles (R. dybowskii, R. rugosa, R. catesbeiana, and Bombina orientalis). Ovarian follicles were collected from each species and manually microdissected to obtain various follicular components: theca-epithelium (THEP) and granulosa cell-enclosed oocyte (GCEO). Subsequent to collection, equal numbers of intact follicles and various follicular components were cultured for 6 hr in the presence of known inducers of steroidogenesis (frog pituitary homogenate [FPH] or 3-iso-butyl-1-methylxanthine [IBMX] + forskolin) or various steroids that serve as substrates for specific steroidogenic enzymes. Following incubation, culture medium was collected and analyzed by radioimmunoassay (RIA). Both FPH and IBMX + forskolin consistently stimulated secretion of androstenedione (AD), testosterone (T), and estradiol (E2) from intact follicles obtained from all four frog species. Additionally, in R. dybowskii, these treatments stimulated secretion of progesterone (P4) and 17α-hydroxyprogesterone (17α-OHP4) into the culture medium. Intact follicles obtained from all species readily converted pregnenolone (P5), P4, and 17α-OHP4 to AD, T, and E2. In contrast GCEO converted P5, P4, and 17α-OHP4 to AD and E2, but not to T. However, AD, but not P5, P4, or 17α-OHP4, was converted to T when cultured in the presence of isolated THEP. The microdissection procedure was also modified to isolate THEP without contaminating granulosa cells. The steroidogenic capacities of “impure” THEP and “pure” theca-epithelium (P-THEP) were then compared. Basal amounts of P4 were produced when P5 was added to P-THEP, whereas significantly higher amounts were produced in the presence of impure THEP. No significant conversion of P5 or P4 to 17α-OHP4 occurred following culture with pure or impure THEP layer. Results suggest that the enzyme activity necessary to metabolize AD T is localized in the THEP, whereas the metabolic capacities to convert P5 AD and T E2 are present in the granulosa cell. Furthermore, the data show that the two-cell model is applicable to other frog species. J. Exp. Zool. 284:91–99, 1999. © 1999 Wiley-Liss, Inc.

Changes in the levels of prostaglandin F2α (PGF2α) and E2 (PGE2) in culture medium during in vitro ovulation of Rana dybowskii follicles were examined. The ovulation was induced by frog pituitary homogenate (FPH) or TPA (12‐O‐tetradecanoylphorbol‐13‐acetate, a protein kinase activator) and the levels of PGs were measured by radioimmunoassay. When the ovarian follicles were cultured, only a few oocytes were ovulated by 12 h, but half of them were ovulated by 24 h in response to FPH, whereas around 30% of oocytes were ovulated by 12 h and maximum ovulation (around 50%) occurred by 24 h in response to TPA. Without any stimulation (control), no ovulation occurred. TPA elevated the level of PGF2α to high levels when compared to control (basal levels), but the increase by FPH was less evident. Likewise, the levels of PGE2 increased markedly in response to TPA, but rather decreased by FPH treatment. Interestingly, PGF2α induced ovulation but PGE2 suppressed FPH‐ or PGF2α‐induced oocyte ovulation. Basal levels of...

The present study was designed to investigate whether noradrenergic neurotransmission regulates the gene expression of gonadotropin-releasing hormone (GnRH) in the preoptic area and GnRH receptor in the pituitary. To this end, N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4, 50 mg/kg), an intraperitoneal (i.p.) injection of selective noradrenergic neurotoxin, was administered 1 h before progesterone (1 mg) treatment in ovariectomized and estradiol-treated prepubertal rats. Treatment with DSP4 effectively blocked the progesterone-induced increase in hypothalamic noradrenaline content, but not dopamine content, indicating that DSP4 selectively inhibits noradrenergic neurotransmission. DSP4 significantly blocked progesterone-induced increase in serum luteinizing hormone (LH) concentrations as well as GnRH release from hypothalamic fragments incubated in vitro. DSP4 concomitantly down-regulated GnRH mRNA levels in the preoptic area, as determined by competitive reverse transcription-polymerase chain reaction. DSP4 also clearly down-regulated progesterone-induced GnRH receptor mRNA levels in the pituitary, whereas it failed to alter LHbeta mRNA levels. In summary, blockade of noradrenergic neurotransmission with DSP4 resulted in profound reductions of hypothalamic GnRH and pituitary GnRH receptor gene expression.

The present study investigated the involvement of the phospholipase C (PLC) and protein kinase C (PKC) signaling pathways during progesterone‐induced meiotic maturation in amphibian (Rana dybowskii) oocytes. Progesterone‐induced germinal vesicle breakdown (GVBD) of oocytes was significantly inhibited by a PKC inhibitor, staurosporine and a PLC inhibitor, U73122, in a dose‐dependent manner. In contrast, U73343, an inactive analogue of U73122, was ineffective in suppressing GVBD. PKC activity in oocytes reached a maximum level at 30 min after progesterone stimulation and this elevated PKC activity was effectively suppressed by U73122 or staurosporine, suggesting that the activation of PKC enzyme is closely linked to PLC signaling during oocyte maturation. In addition, these inhibitors blocked the maturation promoting factor (MPF) activity which appeared in oocytes in response to progesterone, suggesting that PKC activation is an important signal for MPF activity. Therefore, this study demonstrates that the ...

Changes in plasma and pituitary levels of two gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), in male and female Korean frogs (Rana dybowskii and Rana nigromaculata) were examined. Plasma levels of LH and FSH were measured by radioimmunoassay using antibodies raised against bullfrog LH and FSH. In male and female R. dybowskii, plasma concentrations of LH were low in early hibernation (October-January) and increased to high levels by late hibernation, when breeding occurs (late February-early March). Plasma FSH levels were also higher in the breeding period than during hibernation in male and female animals, but absolute levels of FSH were much lower than those of LH. In females, pituitary LH levels were higher in early hibernation, whereas pituitary FSH in females and pituitary LH and FSH in males changed little during hibernation. Plasma LH levels of frogs having uterine eggs or in amplexus were much higher (25 ng/ml) than those of frogs with unovulated follicles (December) (8 ng/ml) or frogs that had already spawned (11 ng/ml). In R. nigromaculata, plasma LH and FSH levels of females collected during hibernation (October-May) were relatively low; however, following hibernation, plasma LH and FSH levels were markedly elevated for a short period. Thus, these animals exhibit a clear gonadotropin surge prior to ovulation and spawning. Soon after spawning, however, gonadotropin levels dropped to basal levels. Following spawning in females, levels of FSH increased steadily and rapid follicle growth occurred until August. By September, FSH had dropped to basal levels. In males, a sharp elevation of LH and FSH levels occurred during the short breeding period followed by a second increase in August, when early stages of spermatogenesis were evident.

Laminin may be involved in uterine re-organization and embryo attachment to the uterine wall during the peri-implantation period. In the present study using a competitive reverse transcription-polymerase chain reaction (RT-PCR), the precise expression patterns of laminin chain (A, B1, and B2)-specific mRNAs were examined in mouse uterine tissues during the peri-implantation period. Although Northern blot hybridization failed to detect laminin A chain mRNA in mouse uterus, RT-PCR analysis showed that laminin A chain mRNA was present even at the lower level compared with B1 and B2 chain mRNA levels. Competitive RT-PCR revealed that ∼3 × 106, 3.6 × 107 , and 4 × 108 copies of A, B1, and B2 chain mRNA transcripts were present in 1 μg of total RNA isolated from the uterus. During pregnancy, the A chain mRNA level was significantly increased only from day 6 after post-hCG when embryo attachment and decidualization started. Elevated level of A chain mRNA was sustained thereafter. Laminin A chain mRNA synthesized at this period was mainly originated from stroma decidual cells. The discrete elevation of laminin A chain mRNA level was also observed after estrogen stimulation in the delayed implantation model. Estrogenic stimulation to ovariectomized, progesterone-treated pregnant mice resulted in about a three-fold increase of laminin A chain mRNA levels. In contrast to A chain mRNA, both B1 and B2 chain mRNA levels were insignificantly altered during the peri-implantation period and delayed implantation by an estrogenic stimulation. Taken together, our results for the first time demonstrate that: (1) laminin A chain mRNA as well as B chain mRNAs is expressed in mouse uterus, (2) its mRNA level is significantly increased along with implantation process, and (3) ovarian steroids, especially estrogen, are likely to be involved in the regulation of laminin gene expression in the uterus. Mol. Reprod. Dev. 48:176–184, 1997. © 1997 Wiley-Liss, Inc.

Effects of gonadotropin and sodium orthovanadate on oocyte maturation, ovulation, prostaglandin, and progesterone synthesis were examined during in vitro culture of Rana ovarian follicles obtained from hibernating animals. Frog pituitary homogenates (FPH, 0.05 gland/ml) effectively induced oocyte maturation (germinal vesicle breakdown, GVBD) and ovulation (> 90%) in ovarian follicles obtained in mid-hibernation (mid-December through late January). In contrast, orthovanadate induced a limited amount ( 90%) of those ovulated in response to FPH had matured. However, the majority of oocytes ovulated (72%) within 6 h of exposure to FPH had intact GVs, indicating that GVBD is also not a prerequisite for ovulation induction in response to FPH. Orthovanadate stimulated PGF2 alpha in a dose dependent manner but failed to stimulate progesterone production whereas FPH stimulated secretion of both PGF2 alpha and progesterone. The amount and time course of PGF2 alpha secretion in response to orthovanadate were similar to results produced with FPH stimulation. Treatment of PGF2 alpha to follicles obtained in late hibernation also accelerated the ovulation of the oocytes. Taken together, the data suggest that orthovanadate enhanced ovulation of immature oocytes was mediated via enhanced PGF2 alpha production and that oocyte maturation is not essential or prerequisite for in vitro oocyte ovulation in Rana.

The steroidogenic acute regulatory (StAR) protein is a key regulator for the steroidogensis in acute response to trophic hormone. A rat complementary DNA of the StAR protein was cloned and its complete nucleotide sequence was determined.2The deduced amino acid sequence of the clone has an additional 86 amino acid stretch at amino terminus when it was compared with those sequences in other species. The other part of the amino acid sequence has 94% identity to mouse StAR protein sequence. Three transcripts (1.3 kb, 1.6 kb, and 3.5 kb) which are hybridizing to the clone were detected in testis, ovary and adrenal gland. When the cDNA was expressed in COS1 cells, 30 kDa and 47 kDa proteins specific to the anti-StAR antibody were detected.

Using three species of Korean frogs (Rana dybowskii, R. rugosa and R. nigromaculata), the annual spermatogenic pattern, the seasonal changes in the steroidogenic competence, and responsiveness of testis to gonadotropins in terms of testosterone secretion in vitro were examined. The spermatogenic pattern of R. dybowskii was classified as a discontinuous type since spermatogenesis stops completely after spawning in late winter (February) until mid‐summer (July). In contrast, the pattern of R. nigromaculata and R. rugosa was classified as a potent continuous type since sperm was always present in the seminiferous tubules all year round. In all three species, the levels of testicular testosterone and that of testosterone secreted by testis following in vitro culture were very low in late summer (August), but increased thereafter until winter (hibernation period). Interestingly, responsiveness of testis in vitro to gonadotropins in terms of testosterone secretion increased markedly in November (early hibernati...

L’article original est publié par The Endocrine Society; The sulfated neurosteroids pregnenolone sulfate (Δ(5)PS) and dehydroepiandrosterone sulfate (DHEAS) are known to play a role in the control of reproductive behavior. In the frog Pelophylax ridibundus, the enzyme hydroxysteroid sulfotransferase (HST), responsible for the biosynthesis of Δ(5)PS and DHEAS, is expressed in the magnocellular nucleus (Mgd) and the anterior preoptic area (Poa), two hypothalamic regions that are richly innervated by GnRH1-containing fibers. This observation suggests that GnRH1 may regulate the formation of sulfated neurosteroids to control sexual activity. Double-labeling of frog brain slices with HST and GnRH1 antibodies revealed that GnRH1-immunoreactive fibers are located in close vicinity of HST-positive neurons. The cDNAs encoding three GnRH receptors (designated riGnRHR-1, -2, and -3) were cloned from the frog brain. Reverse transcription-polymerase chain reaction analyses revealed that riGnRHR-1 is strongly expressed in the hypothalamus and the pituitary while riGnRHR-2 and -3 are primarily expressed in the brain. In situ hybridization histochemistry indicated that GnRHR-1 and GnRHR-3 mRNAs are particularly abundant in Poa and Mgd whereas the concentration of GnRHR-2 mRNA in these two nuclei is much lower. Pulse-chase experiments using tritiated Δ(5)P and DHEA as steroid precursors, and 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as a sulfonate moiety donor, showed that GnRH1 stimulates in a dose-dependent manner the biosynthesis of Δ(5)PS and DHEAS in frog diencephalic explants. Since Δ(5)PS and DHEAS, like GnRH, stimulate sexual activity, our data strongly suggest that some of the behavioral effects of GnRH could be mediated via the modulation of sulfated neurosteroid production.

Laminin is the first extracellular matrix protein that has been shown to be synthesized in preimplantation mouse embryos. In the present study, chain-specific expression patterns of laminin mRNAs were examined by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). During preimplantation mouse embryo development, temporal expression patterns of laminin chain mRNAs were somewhat differential: B1 chain mRNA was first detectable at the late two-cell stage and its level was gradually increased by the blastocyst stage. In contrast, B2 and A chain mRNAs first appeared at the morula and blastocyst stages, respectively. At the blastocyst stage, all of the laminin chain mRNAs were highly detected compared to the earlier stages. When embryos were flushed at the morula stage and cultured in vitro, all laminin chain mRNA levels were decreased or not changed in the process of blastocoele expansion. In contrast, in the in vivo condition where embryos at different stages of blastocyst were flushed at different time points, laminin chain mRNA levels were increased as a function of blastocoele expansion. These changes in laminin mRNAs were parallel with its receptors such as integrin α3 and α6. 3-Isobutyl-1-methylxanthine (IBMX), which is known to be a potent activator of blastocoele expansion and regulates cAMP metabolism, upregulated laminin expression (except B1 chain) in blastocysts cultured in vitro. In vitro cultured embryos normally developed up to the late blastocyst, although their development was delayed compared with the in vivo condition where laminin gene expression was gradually increased as the blastocoele expanded. These results indicate that laminin expression may not be involved directly in the regulation of blastocoele expansion. The uterine environment enclosing the preimplantation embryos appears, therefore, to play an important role in the regulation of laminin gene expression during blastocyst development. © 1996 Wiley-Liss, Inc.

Earlier studies showed that the predominant steroid (estradiol [E 2 ], testosterone [T], progesterone [P 4 ]) secreted by in vitro cultured amphibian ( Rana nigromaculata ) ovarian follicles varied with their size. E 2 was mainly produced by medium-sized follicles, T by intermediate-sized follicles, and P 4 by the largest follicles. Experiments were carried out to ascertain whether the activities of steroidogenic enzymes changed during follicle development and were responsive to gonadotropic stimulation. Enzyme activities were measured indirectly by monitoring conversion of exogenously added substrates to products during in vitro culture of isolated follicles. Different stage follicles were cultured in the presence or absence of frog pituitary homogenate (FPH, 0.1 pituitary/2 ml) and/or various steroid precursors (25-200 ng/2 ml). Amounts of E 2 , T, androstenedione (AD), 17α-hydroxyprogesterone (17α-OHP 4 ), or P 4 secreted into the medium were measured by RIA. Exogenous pregnenolone (P 5 ) was converted to P 4 by all types of follicles in a dose-dependent manner in the absence of FPH. Addition of FPH markedly enhanced medium levels of P 4 in all sized follicles. Highest levels of P 4 were presented in cultures containing the largest follicles. Such follicles were much less efficient than intermediate follicles in metabolizing P 4 to AD or T. EPH suppressed conversion of exogenous 17α-OHP 4 but not androstenedione to testosterone by the largest follicles. Exogenous T was converted to E 2 only by medium-sized follicles and FPH had little or no stimulating or inhibiting effect on this process in either medium- or intermediate-sized follicles. Taken together, the data suggest that (1) the transition from E 2 to T secretion (Class III to IV) is associated with a loss of aromatase activity, (2) the transition from T to P 4 production (Class IV to V) is correlated with a decrease in the activity of enzymes which metabolize P 4 , and (3) activities of steroidogenic enzymes varied in their responsiveness to EPH stimulation.

The steroid secretory activity of cultured ovarian follicles of Rana dybowskii and the activities of relevant steroidogenic enzymes were examined during the natural hibernation period (October-February). Enzyme activities were measured indirectly by monitoring the conversion of exogenous substrates to products by isolated follicles. Follicles were incubated for 6 h in amphibian Ringer in the presence or absence of frog pituitary homogenate (FPH, 0.1 pituitary/2 ml) and/or various steroid precursors. Progesterone (P4), 17α-hydroxyprogesterone (17α-OHP) or testosterone (T) secreted by the follicles into the medium were measured by RIA. In the presence of FPH, high levels of P4 were produced by follicles at the early and mid-hibernation period (695 and 898 pg/follicle, respectively) whereas markedly elevated levels of P4 were produced during late hibernation (1,393 pg/follicle) (just prior to or breeding season, February). In contrast, high levels of T were produced by the follicles early in hibernation (1,206 pg/follicle) while negligible levels were produced in late hibernation (69 pg/follicle). Higher levels of 17α-OHP were produced by follicles at early and mid hibernation (594 and 705 pg/follicle, respectively) than in later hibernation (221 pg/follicle). Addition of exogenous pregnenolone markedly increased P4 levels in a dose-dependent manner when added to follicles at early and late stages of hibernation: FPH addition further enhanced conversion of pregnenolone. Similarly, addition of androstenedione (AD) increased T levels in a dose-dependent manner by these follicles. However, following addition of exogenous P4, less 17α-OHP was produced by follicles collected in late hibernation as compared to those collected earlier. Likewise, addition of 17α-OHP to follicles during late hibernation produced a small increase in T accumulation while a marked increase occurred when follicles were obtained in early hibernation. Taken together, the data indicate that steroidogenesis shifts from T to P4 late in hibernation and this is probably due to decreases in 17α-hydroxylase and C17, 20 -lyase activities. © 1993 Wiley-Liss, Inc.

We previously reported that protein kinase C (PKC) activation induced meiotic maturation (germinal vesicle breakdown, GVBD) of Rana dybowskii follicular oocytes cultured in vitro without hormone treatment. The experiments reported here were carried out to establish whether ovarian follicles ovulated in response to PKC activation during culture. A phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), was used for PKC activation. TPA addition (10 microM) to cultured ovarian fragments induced ovulation and maturation of the oocytes similar to that seen following addition of frog pituitary homogenate (FPH, 0.05 pituitary/ml) or progesterone (0.5 microgram/ml). Such changes were not observed when ovarian fragments were treated with inactive phorbol ester. The time course of TPA-induced ovulation was similar to that produced by FPH-stimulated ovulation. Both TPA- and FPH-stimulated ovulation and maturation were blocked by treatment with cycloheximide, forskolin (an adenylate cyclase stimulator), and 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7; a PKC inactivator). FPH treatment markedly increased progesterone levels in the medium during ovarian fragment culture whereas TPA treatment failed to elevate progesterone levels. Thus, TPA treatment mimics FPH and progesterone in inducing ovulation and meiotic maturation in cultured amphibian ovarian fragments. The data strongly suggest that PKC plays an important role in regulating ovulation as well as in modulating amphibian oocyte maturation during follicular differentiation.