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G-protein-coupled receptors (GPCR) are now regarded as being able to acquire heterodimer conformations affecting their pharmacology, signaling and trafficking. In co-immunoprecipitation studies using differentially epitope-tagged receptors, we herein provide direct evidence for heterodimerization of human neurotensin type 1 receptor (hNTR1) and type 2 receptor (hNTR2). Using chimeric constructs, we also identified the hNTR2 transmembrane 2 (TM2) to TM4 region as crucial for the formation of the dimerization interface. At the functional level, we demonstrated that the co-expression of hNTR2 suppressed hNTR1-mediated adenylate cyclase/cAMP and phospholipase C activation. Finally, confocal microscopy revealed that whereas tagged hNTR1 expressed alone were localized to the plasma membrane, co-expression of hNTR2 caused the retention of hNTR1 in sub-cellular compartments, indicating that heterodimerization with hNTR2 interferes with the proper recruitment of hNTR1 to the plasma membrane. Overall, this study proposes a novel function of NTR2 in the regulation of NTR1 activity.

Kisspeptin and its receptor GPR54 play important roles in mammalian reproduction and cancer metastasis. Because the KiSS and GPR54 genes have been identified in a limited number of vertebrate species, mainly in mammals, the evolutionary history of these genes is poorly understood. In the present study, we have cloned multiple forms of kisspeptin and GPR54 cDNAs from a variety of vertebrate species. We found that fish have two forms of kisspeptin genes, KiSS-1 and KiSS-2, whereas Xenopus possesses three forms of kisspeptin genes, KiSS-1a, KiSS-1b, and KiSS-2. The nonmammalian KiSS-1 gene was found to be the ortholog of the mammalian KiSS-1 gene, whereas the KiSS-2 gene is a novel form, encoding a C-terminally amidated dodecapeptide in the Xenopus brain. This study is the first to identify a mature form of KiSS-2 product in the brain of any vertebrate. Likewise, fish possess two receptors, GPR54-1 and GPR54-2, whereas Xenopus carry three receptors, GPR54-1a, GPR54-1b, and GPR54-2. Sequence identity and genome synteny analyses indicate that Xenopus GPR54-1a is a human GPR54 ortholog, whereas Xenopus GPR54-1b is a fish GPR54-1 ortholog. Both kisspeptins and GPR54s were abundantly expressed in the Xenopus brain, notably in the hypothalamus, suggesting that these ligand-receptor pairs have neuroendocrine and neuromodulatory roles. Synthetic KiSS-1 and KiSS-2 peptides activated GPR54s expressed in CV-1 cells with different potencies, indicating differential ligand selectivity. These data shed new light on the molecular evolution of the kisspeptin-GPR54 system in vertebrates.

Rana ovarian follicles at different developmental stages were obtained from frogs collected at different seasons. Isolated follicles were sorted into five different classes (Class I–V) by size and cytoplasmic pigmentation. They were incubated for 6 hours in amphibian Ringer in the presence or absence of frog pituitary homogenate (FPH, 0.05 pituitary/ml) and changes in steroid production by follicles were assessed by measuring estradiol (E2), testosterone (T), and progesterone (P4) in media and follicles by radioimmunoassay. Small follicles (size range 0.22–0.7 mm) secreted very low or non-detectable levels of the three steroids in the presence or absence of FPH. Medium-sized vitellogenic follicles (0.70–1.11 mm) actively secreted all the three types of steroids tested (E2, P4, and T). Larger follicles (1.15–1.58 mm), isolated during September–February, secreted much more T than E2 and P4. In contrast, fully grown follicles (> 1.50 mm) collected late in the hibernation period secreted more P4 and less T than the large follicles and very low levels of E2; however, follicles (Class V) collected in breeding season (May) secreted solely P4 and negligible amounts of T and E2. All types of follicles produced very low levels of the steroids without FPH stimulation. Intrafollicular levels of steroids essentially reflected those which accumulated in the medium. Oocyte responsiveness to exogenous hormone, as evidenced by germinal vesicle breakdown (GVBD), increased as the breeding season approached. The data indicate that only the medium-sized follicles produced considerable amounts of E2 while larger follicles produced mainly T and full-grown follicles collected during breeding season produced only P4.

The present study was undertaken to elucidate the mechanism of nuclear membrane dissolution (NMD) in puromycin-treated mouse oocytes. Treatment of germinal vesicle breakdown (GVBD) oocytes with puromycin (50 micrograms/ml) induced chromosome decondensation with formation of a polar body; these are designated nuclear membrane (NM) oocytes. After withdrawal of puromycin, NM oocytes underwent NMD (approximately 70%) during a 12-h culture period. Either dibutyryl cyclic AMP (dbcAMP, 25-100 micrograms/ml) or isobutylmethylxanthine (IBMX, 0.1-1.0 mM) inhibited the process of NMD in a dose-dependent manner, suggesting the involvement of cAMP in the process of NMD. To determine which protein(s) participated in the transition from interphase to metaphase II during NMD, NM oocytes were labeled with [35S]methionine, and one- and two-dimensional gel electrophoresis were performed. Although the synthesis of stage-specific proteins during NMD was not found, two specific proteins of Mr 27,000 and 46,000, which were synthesized at interphase following removal of puromycin, were modified during NMD. Phosphatase treatment and 32PO4-labeling experiments indicated that phosphorylation was responsible for these modifications, which were inhibited by either dbcAMP or IBMX. Therefore, it appears that phosphorylation of specific proteins may play an important role in the transition from interphase to metaphase II.

The Glu/Asp(7.32) residue in extracellular loop 3 of the mammalian type-I gonadotropin-releasing hormone receptor (GnRHR) interacts with Arg(8) of GnRH-I, conferring preferential ligand selectivity for GnRH-I over GnRH-II. Previously, we demonstrated that the residues (Ser and Pro) flanking Glu/Asp(7.32) also play a role in the differential agonist selectivity of mammalian and non-mammalian GnRHRs. In this study, we examined the differential antagonist selectivity of wild type and mutant GnRHRs in which the Ser and Pro residues were changed. Cetrorelix, a GnRH-I antagonist, and Trptorelix-2, a GnRH-II antagonist, exhibited high selectivity for mammalian type-I and non-mammalian GnRHRs, respectively. The inhibitory activities of the antagonists were dependent on agonist concentration and subtype. Rat GnRHR in which the Ser-Glu-Pro (SEP) motif was changed to Pro-Glu-Val (PEV) or Pro-Glu-Ser (PES) had increased sensitivity to Trptorelix-2 but decreased sensitivity to Cetrorelix. Mutant bullfrog GnRHR-1 with the SEP motif had the reverse antagonist selectivity, with reduced sensitivity to Trptorelix-2 but increased sensitivity to Cetrorelix. These findings indicate that the residues flanking Glu(7.32) are important for antagonist as well as agonist selectivity.

Kisspeptin and its receptor, GPR54, play important roles in mammalian reproduction and cancer development. However, little is known about their function in nonmammalian species. In the present study, we have isolated the cDNA encoding the kisspeptin receptor, GPR54, from the bullfrog, Rana catesbeiana. The bullfrog GPR54 (bfGPR54) cDNA encodes a 379-amino acid heptahelical G protein-coupled receptor. bfGPR54 exhibits 45-46% amino acid identity with mammalian GPR54s and 70-74% identity with fish GPR54s. RT-PCR analysis showed that bfGPR54 mRNA is highly expressed in the forebrain, hypothalamus and pituitary. Upon stimulation by synthetic human kisspeptin-10 with Phe-amide residue at the C-terminus (h-Kiss-10F), bfGPR54 induces SRE-luc activity, a PKC-specific reporter, evidencing the PKC-linked signaling pathway of bfGPR54. Using a blast search, we found a gene encoding a kisspeptin-like peptide in Xenopus. The C-terminal decapeptide of Xenopus kisspeptin shows higher amino acid sequence identity to fish Kiss-10s than mammalian Kiss-10s. A synthetic Xenopus kisspeptin peptide (x-Kiss-12Y) showed a higher potency than mammalian Kiss-10s in the activation of bfGPR54. This study expands our understanding of the physiological roles and molecular evolution of kisspeptins and their receptors.

Purpose The primary objective of this study was to examine the changes of basal cortisol and DHEA levels present in saliva and serum with age, and to determine the correlation coefficients of steroid concentrations between saliva and serum. The secondary objective was to obtain a standard diurnal rhythm of salivary cortisol and DHEA in the Korean population. Materials and Methods For the first objective, saliva and blood samples were collected between 10 and 11 AM from 359 volunteers ranging from 21 to 69 years old (167 men and 192 women). For the second objective, four saliva samples (post-awakening, 11AM, 4PM, and bedtime) were collected throughout a day from 78 volunteers (42 women and 36 men) ranging from 20 to 40 years old. Cortisol and DHEA levels were measured using a radioimmunoassay (RIA). Results The morning cortisol and DHEA levels, and the age-related steroid decline patterns were similar in both genders. Serum cortisol levels significantly decreased around forty years of age (p < 0.001, when compared with people in their 20s), and linear regression analysis with age showed a significant declining pattern (slope = -2.29, t = -4.297, p < 0.001). However, salivary cortisol levels did not change significantly with age, but showed a tendency towards decline (slope = -0.0078, t = -0.389, p = 0.697). The relative cortisol ratio of serum to saliva was 3.4-4.5% and the ratio increased with age (slope = 0.051, t = 3.61, p < 0.001). DHEA levels also declined with age in saliva (slope = -0.007, t = -3.76, p < 0.001) and serum (slope = -0.197 t = -4.88, p < 0.001). In particular, DHEA levels in saliva and serum did not start to significantly decrease until ages in the 40s, but then decreased significantly further at ages in the 50s (p < 0.001, when compared with the 40s age group) and 60s (p < 0.001, when compared with the 50 age group). The relative DHEA ratio of serum to saliva was similar throughout the ages examined (slop = 0.0016, t = 0.344, p = 0.73). On the other hand, cortisol and DHEA levels in saliva reflected well those in serum (r = 0.59 and 0.86, respectively, p < 0.001). The highest salivary cortisol levels appeared just after awakening (about two fold higher than the 11 AM level), decreased throughout the day, and reached the lowest levels at bedtime (p < 0.001, when compared with PM cortisol levels). The highest salivary DHEA levels also appeared after awakening (about 1.5 fold higher than the 11AM level) and decreased by 11AM (p < 0.001). DHEA levels did not decrease further until bedtime (p = 0.11, when compared with PM DHEA levels). Conclusion This study showed that cortisol and DHEA levels change with age and that the negative slope of DHEA was steeper than that of cortisol in saliva and serum. As the cortisol and DHEA levels in saliva reflected those in serum, the measurement of steroid levels in saliva provide a useful and practical tool to evaluate adrenal functions, which are essential for clinical diagnosis.

In mammals, apelin and its G protein-coupled receptor, APJ, regulate blood pressure, intake of food and water, and cardiac contractility. In this study, we report the cloning and functional characterization of APJ in the bullfrog, Rana catesbeiana . Bullfrog APJ (bfAPJ) cDNA contains an open reading frame of 1083 nucleotides encoding a protein of 360 amino acid residues. Sequence alignment reveals 75% amino acid identity with Xenopus , 63% identity with zebrafish and 40–42% identity with mammalian APJs. RT-PCR analysis and tissue binding assay reveal high expression of bfAPJ mRNA in the brain, particularly in the hypothalamus, and moderate expression in the pituitary, testis, adrenal gland and lung. Whereas [pGlu 1 ]apelin-13 did not induce CRE-luc (protein kinase A-specific reporter) and SRE-luc (protein kinase C-specific reporter) activity in cells expressing bfAPJ, this apelin-13 decreased forskolin-induced CRE-luc activity and cAMP accumulation in a pertussis toxin-sensitive manner. This study indicates that bfAPJ may couple to G i/o . [Pro 1 ]apelin-13, a synthetic apelin based on the sequence of the putative apelin gene from many non-mammalian species, activates bfAPJ with 5–10-fold greater sensitivity/affinity than mammalian apelin-13. Collectively, this study expands our understanding of the physiological roles of this receptor system in non-mammalian species.

The superfamily of G protein-coupled receptors (GPCRs) is the largest and most diverse group of membrane-spanning proteins. It plays a variety of roles in pathophysiological processes by transmitting extracellular signals to cells via heterotrimeric G proteins. Completion of the human genome project revealed the presence of approximately 168 genes encoding established nonsensory GPCRs, as well as 207 genes predicted to encode novel GPCRs for which the natural ligands remained to be identified, the so-called orphan GPCRs. Eighty-six of these orphans have now been paired to novel or previously known molecules, and 121 remain to be deorphaned. A better understanding of the GPCR structures and classification; knowledge of the receptor activation mechanism, either dependent on or independent of an agonist; increased understanding of the control of GPCR-mediated signal transduction; and development of appropriate ligand screening systems may improve the probability of discovering novel ligands for the remaining orphan GPCRs.

There are two forms of gonadotrophin-releasing hormone (GnRH), GnRH-I and GnRH-II, in the vertebrate brain. Both GnRH-I and GnRH-II are thought to interact with the type-I GnRH receptor (GnRHR). The present study attempted to demonstrate whether GnRH-I and GnRH-II induce differential desensitisation of GnRHR and to identify the motif involved. Time course inositol phosphate (IP) accumulation assay reveals that, in cells expressing the wild-type rat GnRHR, GnRH-I induced continuous increase in IP production, whereas GnRH-II-induced IP production rate at later time points (30-120 min after ligand treatment) became attenuated. However, in cells expressing the mutant receptor in which the Ser-Glu-Pro (SEP) motif in extracellular loop 3 was replaced by Pro-Glu-Val (PEV), IP accumulation rates at later time points were more decreased by GnRH-I than GnRH-II. Ca 2+ responses to repetitive GnRH applications reveal that GnRH-II desensitised the wild-type receptor faster than GnRH-I, whereas the opposite situation was observed in the PEV mutant. In addition, cell surface loss of GFP-tagged wild-type receptor was more facilitated by GnRH-II than GnRH-I, whereas that of the GFP-tagged PEV mutant receptor was more enhanced by GnRH-I than GnRH-II. The present study indicates that the SEP motif is potentially responsible for ligand species-dependent receptor desensitisation. Together, these results suggest that GnRH-I and GnRH-II may have different effects on mammalian type-I GnRHR via modulation of desensitisation rates.

The superfamily of G protein-coupled receptors (GPCRs) is the largest and most diverse group of membrane-spanning proteins. It plays a variety of roles in pathophysiological processes by transmitting extracellular signals to cells via heterotrimeric G proteins. Completion of the human genome project revealed the presence of approximately 168 genes encoding established nonsensory GPCRs, as well as 207 genes predicted to encode novel GPCRs for which the natural ligands remained to be identified, the so-called orphan GPCRs. Eighty-six of these orphans have now been paired to novel or previously known molecules, and 121 remain to be deorphaned. A better understanding of the GPCR structures and classification; knowledge of the receptor activation mechanism, either dependent on or independent of an agonist; increased understanding of the control of GPCR-mediated signal transduction; and development of appropriate ligand screening systems may improve the probability of discovering novel ligands for the remaining orphan GPCRs.

International audience; Neurotensin (NT) is a tridecapeptide that functions as a neurotransmitter and neuromodulator in the nervous system. To date, three different types of NT receptor (NTR), NTR1, NTR2 and NTR3, have been identified only in mammalian species. In the present study we isolated the cDNAs for an NTR1 and a novel NTR in the bullfrog brain, designated bfNTR1 and bfNTR4 respectively. bfNTR1 and bfNTR4 encode 422- and 399-amino acid residue proteins respectively. bfNTR1 has a 64% amino acid identity with mammalian NTR1, and 34-37% identity with mammalian NTR2. bfNTR4 exhibits 43% and 45-47% identity with mammalian NTR1 and NTR2 respectively. Both receptors are mainly expressed in the brain and pituitary. bfNTR1 triggers both CRE-luc, a protein kinase A (PKA)-specific reporter, and c-fos-luc, a PKC-specific reporter, activities, indicating that bfNTR1 can activate PKA- and PKC-linked signaling pathways. However, bfNTR4 appears to be preferentially coupled to the PKA-linked pathway as it induces a higher CRE-luc activity than c-fos-luc activity. bfNTRs exhibit different pharmacological properties as compared with mammalian NTRs. Mammalian NTR1 but not NTR2 responds to NT, whereas both bfNTR1 and bfNTR4 show a high sensitivity to NT. SR 48692 and SR 142948A, antagonists for mammalian NTR1 but agonists for mammalian NTR2, function as antagonists for both bfNTR1 and bfNTR4. In conclusion, this report provides the first molecular, pharmacological and functional characterization of two NTRs in a non-mammalian vertebrate. These data should help to elucidate the phylogenetic history of the G protein-coupled NTRs in the vertebrate lineage as well as the structural features that determine their pharmacological properties.

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.

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 aim of our in vitro experiments was to study the role of oxytocin (OT), cAMP/protein kinase A (PKA), and mitogen-activated protein kinase (ERKs MAP-kinase) in the control of ovarian cell functions as well as the role of PKA and MAPK in mediating OT effects on these processes. The whole porcine ovarian follicles were cultured in the presence or absence of OT (1, 10, 100 ng/ml), PKA inhibitor Rp-cAMPS (10 nM), MAP-kinase inhibitor PD98059 (1 microg/ml), or their combination. The release of prostaglandins F (PGF) and E (PGE) were determined by RIA, PKA (alpha-cat subunit), the proliferation-associated peptide PCNA and ERK-1, -2 expression in cell lyzates were analysed by Western-blotting. OT stimulated the release of PGF and PGE, and accumulation of PKA, ERK-1/-2, and PCNA in cell lysate. PD98059 decreased the basal PGF and PGE output, as well as reduced both ERK-1 and ERK-2 accumulation in cell lysates. Rp-cAMPS decreased PKA accumulation in cell lysates. Rp-cAMPS prevented the OT-induced stimulation of PKA, ERK-1, ERK-2, PGF, and PGE, PD98059 did so for PKA, PGF, and PGE. However, PD98059 reduced either basal or OT-induced p-ERK level. OT-stimulated PCNA accumulation was only slightly modified by these blockers. These observations suggest that OT, PKA, and ERKs MAPK can be involved in the control of PGs release and proliferation of ovarian cells. The influence of OT on both PKA and MAPK, and the ability of PKA and MAPK blockers to prevent completely or partially OT effects suggest, that effects of OT on PGF and PGE can be mediated by both PKA and MAPK. The role of MAPK and PKA in mediating the proliferative effects of OT seems to be minor assuming the involvement of other intracellular messengers.

Cadmium (Cd) is one of the environmental pollutants affecting various tissues and organs including testis. Harmful effect of Cd in testis is known to be germ cell degeneration and impairment of testicular steroidogenesis. Animals treated with high doses of Cd (0.2 and 0.3 mg/100 g BW) showed a significant decrease in serum testosterone (T) level, but a significant induction of testicular lipid peroxidation levels. TUNEL assay showed that low doses of Cd (0.13 and 0.15 mg/100 g BW) exhibited typical characteristics of apoptosis while high doses of Cd caused more necrosis than apoptosis. In contrast, supplementation with ascorbic acid reduced testicular lipid peroxidation levels. Ascorbic acid supplementation restored testicular 3β-hydroxysteroiddehydrogenase (HSD) and 17β-HSD enzyme activities, 3β-HSD and cytochrome P450 side chain cleavage (P450scc) mRNA levels and serum T concentration to normal in Cd-administered rats. Moreover, administration of ascorbic acid prevented germ cell apoptosis as demonstrated by the reduced number of TUNEL-positive cells in germinal epithelium and inhibited Cd-induced necrosis. These results indicate that ascorbic acid have protective roles in vivo on the Cd-induced overall testicular damage including impaired steroidogenesis and germ cell death possibly through scavenging the reactive oxygen species generated by Cd administration.

Recently, we identified three types of non-mammalian gonadotropin-releasing hormone receptors (GnRHR) in the bullfrog (designated bfGnRHR-1-3), and a mammalian type-II GnRHR in green monkey cell lines (denoted gmGnRHR-2). All these receptors responded better to GnRH-II than GnRH-I, while mammalian type-I GnRHR showed greater sensitivity to GnRH-I than GnRH-II. In the present study, we designed new GnRH-II analogs and examined whether they activated or inhibited non-mammalian and mammalian type-II GnRHRs. [D-Ala6]GnRH-II, with D-Ala substituted for Gly6 in GnRH-II, increased inositol phosphate (IP) production in cells stably expressing non-mammalian GnRHRs more effectively than native GnRH-II. However, it exhibited lower activity for mammalian type-I GnRHR than GnRH-I itself. Trptorelix-1, a GnRH-II antagonist, inhibited GnRH-induced IP production in cells expressing non-mammalian GnRHRs more effectively than Cetrorelix, a GnRH-I antagonist. Trptorelix-1, however, had lower potency for mammalian type-I GnRHR than Cetrorelix. Ligand-receptor binding assays revealed that [D-Ala6]GnRH-II and Trptorelix-1 have higher affinities for non-mammalian GnRHRs but lower affinities for mammalian type-I GnRHR than GnRH-II and Cetrorelix, respectively. Moreover, [D-Ala6]GnRH-II and Trptorelix-1 had a higher affinity for gmGnRHR-2 than GnRH-II and Cetrorelix, respectively. These results indicate that [D-Ala6]GnRH-II and Trptorelix-1 are highly effective agonist and antagonist, respectively, for non-mammalian and type-II mammalian GnRHRs.

We have cloned and characterized a full-length cDNA of androgen receptor (AR) from the testis of bullfrog, Rana catesbeiana . The cDNA contains an open reading frame of 2328 nucleotides encoding a protein of 776 amino acid residues. The bullfrog AR shows high homology with ARs from other species in its amino acid sequence. Its overall homology with those of African clawed frog, Japanese eel and human is 70, 53, and 63%, respectively. As expected, the N-terminal domain shows much less homology (30–59%) than both DNA-binding domain (85–92%) and ligand binding domain (80–89%). Northern blot analysis detected the bullfrog AR message as a single transcript of around 9 kb only in the testis. However, RT-PCR analysis revealed that AR mRNA is also expressed in other tissues although the levels are very low compared to that in the testis. Western blot analysis of whole tissue extracts showed the presence of AR protein in fore brain, heart, and testis. The AR gene is present as a single copy in bullfrog based on Southern blot analysis of genomic DNA. Altogether, the results suggest that the bullfrog AR is evolutionary conserved and may have functions similar to those shown in other species.

Growth hormone (GH), prostaglandins F (PGF) and prostaglandins E (PGE) are important regulators of ovarian function. Therefore, interrelationships between GH and these substances and their intracellular mechanisms might be of physiological significance in the ovary. The aims of this study on cultured porcine ovarian granulosa cells were to determine the effect of GH on the secretion of oxytocin (OT), PGF and PGE and whether MAP kinase could be involved in the mediation of GH action. Experiments were carried out with cultured porcine granulosa cells to investigate the effects of exogenous pGH (1-100 ng/ml) on the expression of MAP kinase (ERK-1, -2) and of PGH (1-100 ng/ml) and the MAP kinase blocker PD 98059 (1 microg/ml) on the secretion of PGF, PGE and OT. The cellular content of ERK-1 and -2 was analyzed by Western immunoblotting and immunocytochemistry, whilst PGF, PGE and OT accumulation in the medium was measured by RIA. Addition of GH to culture medium significantly altered the pattern of ovarian ERK MAP kinase on SDS-PA gels: the 44 and 42 kDa bands were reduced and additional 50 and 48 kDa bands appeared. Moreover, there was an increase in the percentage of cells containing ERK MAP kinase. GH stimulated the secretion of PGF (at a concentration of 1 ng GH per ml medium) and OT (100 ng GH per ml), but not PGE. The MAP kinase blocker alone did not affect PGF, PGE and OT secretion but did prevent the stimulatory effects of GH on PGF and induced stimulatory action of GH (10 ng/ml) on PGE. GH-stimulated OT secretion was unaffected. These observations confirm the role of GH in regulating porcine ovarian PGF, PGE and OT secretion and the presence of ERK MAP kinase in porcine granulosa cells. Furthermore, our studies demonstrate that MAP kinase-dependent intracellular mechanisms are dependent on GH, and that these mechanisms are involved in the mediation of GH action on ovarian PGF and PGE but not OT secretion.

Orphan nuclear receptors belong to the nuclear receptor superfamily of liganded transcription factors, whose ligands either do not exist or remain to be identified. We report here the cloning and characterization of the chicken orphan nuclear receptor, cTR2 (chicken testicular receptor 2). The cTR2 gene encodes a protein of 569 amino acids which shows approximately 72% overall identity with TR2 (NR2C1) and 95% identity in the DNA-binding domain (DBD). The cTR2 gene is expressed in almost all adult tissues and embryonic stages examined unlike its mammalian relative TR2, which is specifically expressed in testis. Electrophoretic mobility shift assays demonstrate that cTR2 binds the canonical direct repeat DNA recognition sequences spaced by one, four, and five nucleotides (DR1, DR4, and DR5), and in consistence with the results with canonical DNA-binding sequences, cTR2 forms specific DNA-protein complex with chicken phenobarbital response elements containing DR4 motifs. Both in vitro and in vivo interaction studies demonstrate that cTR2 forms homodimer. Moreover, transient transfection studies reveal its capability to transactivate canonical DR1, DR4, and DR5 sequences and the constitutive activity of cTR2 is mapped to the N-terminal region of this orphan receptor. Finally, cTR2 represses transactivation of estrogen receptor in a dose-dependent manner.

Recently, certain environmental endocrine disrupters have shown to act as antiandrogens. This suggests that environmental antiandrogens may also be crucial contributors to the increasing incidence of male reproductive abnormalities, requesting the screening and classification of antiandrogenic chemicals. Here, we report the development of a rapid, simple and effective yeast detection system for androgenic and antiandrogenic compounds, which is based on the yeast two-hybrid protein interaction. A yeast strain, ARhLBD-ASC1, was established by co-transformation of yeast cells harboring a lacZ reporter plasmid with two vectors expressing each of LexA fused hinge-ligand binding domain (hLBD) of androgen receptor (AR) and B42 fused ASC-1 that interacts with the AR-hLBD in an androgen-dependent manner. In this yeast strain, androgens, but not other hormones, strongly stimulated the beta-galactosidase activity in a dose-dependent manner. The AR antagonists flutamide, cyproterone acetate and spironolactone, and environmental antiandrogens p,p'-DDE and vinclozolin all inhibited the response of the yeast cells to 10 nM testosterone, qualitatively similar to their inhibition reported in mammalian cell systems. Furthermore, the bioassay can be performed with the simple X-gal staining on microtiter plates, suggesting this system as a powerful tool for practical and efficient screening of environmental compounds for their androgenic and antiandrogenic activities.

A novel inhibitor of apoptosis protein family member termed SIX was identified in Xenopus containing a single baculoviral IAP repeat (BIR) domain and no COOH-terminal RING finger domain. It exhibited striking amino acid sequence similarity with human survivin, mouse TIAP, and recently found Xenopus survivin, especially a part of BIR domain was highly conserved. Interestingly, SIX interacted with RXRα through the AF2 domain in the absence of ligand, which was weakened when the ligand was present. Northern blot analysis demonstrated that SIX mRNA was not detectable in adult with exception of the ovary and testis, and whole-mount in situ hybridization and Northern blot analyses revealed strong and homogeneous expression of SIX in the developing oocytes. In the embryos, the expression of SIX was observed in the animal hemisphere from one-cell to yolk plug stages and high level of expression was detected in the future brain and dorsal region of the neural tube at the neurula stage and early tail-bud stage. These results strongly support the fact that survivin is evolutionarily conserved in structure and SIX is likely to be the Xenopus counterpart of human and mouse survivin.

We previously demonstrated the presence of three distinct types of the gonadotropin-releasing hormone receptor (GnRHR) in a bullfrog (denoted bfGnRHR-1, bfGnRHR-2, and bfGnRHR-3). The bfGnRHRs exhibited differential tissue distribution and ligand selectivity. In the present study, we demonstrated the desensitization and internalization kinetics of these receptors in both transiently-transfected HEK293 cells and retrovirus-mediated stable cells. The time-course accumulation of the inositol phosphate in response to GnRH revealed that bfGnRHR-1 and -2 were rapidly desensitized, whereas bfGnRHR-3 was slowly desensitized. A comparison of the internalization kinetics revealed the most rapid rate and highest extent of internalization of bfGnRHR-2 among the three receptors. Interestingly, the mechanisms that underlie the receptor internalization appear to differ from each other. Internalization of bfGnRHR-1 was dependent on both dynamin and beta-arrestin, whereas those of bfGnRHR-2 and -3 were dependent on dynamin, but not on arrestin. These results, therefore, suggest that differential regulatory mechanisms for desensitization and internalization of the GnRHR are involved in diverse cellular and physiological responses to GnRH stimulation.

Previously, we observed that 70-kDa ribosomal protein S6 kinase (p70(s6k)) plays an essential role during the early phase of oocyte maturation in Rana dybowskii. To investigate further the early signal transduction components involved in this process, the possible role of phosphatidylinositol-3 kinase (PI3 kinase) during oocyte maturation was examined. Progesterone-induced oocyte maturation was significantly inhibited by wortmannin and LY294002, specific inhibitors of PI3 kinase. In contrast, protein kinase C activator 12-0-tetradecanoylphorbol-13-acetate-induced oocyte maturation was not inhibited by wortmannin. Protein synthesis was also significantly suppressed by wortmannin treatment during oocyte maturation. Moreover, PI3 kinase inhibitor suppressed progesterone-induced phosphorylation of S6 kinase in a dose-dependent manner. Likewise, PI3 kinase inhibitors significantly inhibited the phosphorylation of mitogen-activated protein (MAP) kinase which was increased during oocyte maturation. Finally, progesterone-induced H1 kinase activity was also inhibited by PI3 kinase inhibitors in a dose-dependent manner. Taken together, these results suggest that PI3 kinase is an initial component of the signal transduction pathway which precedes p70(s6k), MAP kinase, and MPF production during progesterone-induced maturation of amphibian oocyte.

We have identified and characterized a new amphibian orphan member of the nuclear receptor superfamily and termed it FOR1 (farnesoid X receptor (FXR)-like OrphanReceptor) because it shares the highest amino acid identity with the mammalian FXR. We also identified a variant of FOR1, called FOR2, which has 15 additional C-terminal amino acids. Both variants include an unusual insertion of 33 amino acids in the helix 7 region of the canonical ligand binding domain sequence, suggesting a unique structure for FOR. Northern blot analysis demonstrates that theFOR gene is highly expressed in adult and tadpole liver, kidney, and tail bud stage of the embryo. Detailed expression analysis using in situ hybridization indicates that FOR expression is first detectable at stage 30/31 in the presumptive liver region lasting until stage 41 with a peak level evident at stage 35/36. FOR forms heterodimeric complexes with retinoid X receptor (RXR) as demonstrated by biochemical and mammalian two-hybrid approaches. Gel mobility shift assays demonstrate that FORs form specific DNA-protein complexes on an FXR binding element consisting of an inverted repeat DNA element with 1 nucleotide spacing (IR1) from the phospholipid transfer protein gene promoter. Finally, although FORs do not exhibit constitutive transcriptional activity, frog gallbladder extract significantly augments the transcriptional activities of FORs.

The aims of this study on porcine ovarian granulosa cells were to examine the effect of GH on oxytocin (OT), IGF-I and IGF-I receptors, IGF-binding protein-3 (IGFBP-3), progesterone and prostaglandin E (PGE), as well as to determine whether IGF-I and/or OT may be mediators of GH action. The cells were cultured either with porcine GH (pGH) (1 ng/ml to 10 microg/ml or 100 ng/ml only), antiserum against IGF-I (0.1%), antiserum against OT (0.1%) or a combination of GH (10 ng/ml) with antiserum against IGF-I or antiserum against OT (0.1%). The secretion of IGF-I, OT, IGFBP-3, progesterone and PGE was determined using RIA/IRMA, whilst the IGF-I binding sites were measured using a radioreceptor assay. It was observed that pGH increased the secretion of IGF-I and the abundance of IGF-I binding sites in granulosa cells. Furthermore, GH inhibited OT release, stimulated progesterone and PGE output, but had no significant effect on IGFBP-3 secretion. Immunoneutralization of IGF-I by antiserum against IGF-I inhibited PGE secretion, but it did not influence progesterone or IGFBP-3 secretion. Binding of OT by antiserum suppressed IGFBP-3, PGE, but not progesterone secretion. Neither immunoneutralization of IGF-I nor OT substantially prevented the effects of GH on progesterone, IGFBP and PGE. These observations demonstrate the involvement of GH, IGF-I and OT in the control of porcine ovarian secretory activity and the ability of GH to regulate IGF-I and OT production and IGF-I reception. Nevertheless, lack of correlation between the effects of GH, antiserum against IGF-I and antiserum against OT, as well as the inability of blockade of IGF-I or OT to prevent the effects of GH, suggests that IGF-I and OT, despite their dependence on GH, do not mediate GH action on ovarian cells.

The aim of our in vitro experiments was to examine if IGF binding protein (IGFBP)-3 is involved in control of bovine ovarian secretory activity. For this purpose we performed the transfection of bovine granulosa cells with cDNA sense and antisense constructs increasing or inhibiting IGFBP-3 synthesis. The release of IGFBP-3, progesterone, oxytocin, IGF-I and prostaglandins F (PGF) and E (PGE) by control and transfected cells was compared. The transfected ovarian cells were cultured with and without bLH (100 ng/ml), bGH (100 ng/ml), IGF-I (10 ng/ml), oxytocin (10 ng/ml) and oestradiol-17beta (100 ng/ml). The concentration of IGFBP-3 produced was assessed using ligand and western blotting and secretion of progesterone, oxytocin, IGF-I, PGF and PGE was evaluated using RIA/IRMA techniques. Transfection of cells with the sense IGFBP-3 cDNA construct resulted in the expected increase in IGFBP-3 release, whereas the antisense IGFBP-3 construct induced the expected reduction in IGFBP-3 output. The granulosa cells transfected to overexpress IGFBP-3 had an increase in IGF-I, PGF and PGE release, and a decrease in basal and hormone- or growth factor-induced accumulation of progesterone and oxytocin. The granulosa cells transfected to have reduced IGFBP-3 expression gave primarily significant opposite findings. The present results suggest the involvement of IGFBP-3 in control of bovine ovarian steroid, peptide hormone, growth factor and prostaglandin release. IGFBP-3 is a physiological stimulator of IGF-I and prostaglandin release and an inhibitor of steroid and peptide hormone output.

The effect of transfection with insulin-like growth factor binding protein (IGFBP) sense and antisense cDNA constructs on IGFBP synthesis by cultured bovine and porcine ovarian granulosa cells was studied. Transfection of bovine cells with sense cDNA construct resulted in an increase in basal, GH-, LH-, estradiol- and IGF-I-induced IGFBP-3, IGF-I, PGF and PGE release, and a decrease in P and OT secretion. Treatment with the IGFBP-3 antisense construct resulted in a reduction in IGFBP-3 output. Transfection of porcine granulosa cells with an IGFBP-4 cDNA construct increased the IGFBP-4 and IGF-I production, and decreased P, OT and IGFBP-3 release in the absence or presence of hormones and growth factor. These results suggest that IGFBP-3, IGFBP-4 are potent regulators of the secretory activity of ovarian cells. IGFBPs or an alteration of their production may be useful for controlling ovarian function.

This report is a brief review of our experience in developing fast, sensitive and inexpensive methods for evaluating the biological activity of hormones, growth factors, their analogues and drugs, using cell cultures. It demonstrates that the analysis of parameters such as proliferation, apoptosis, meiosis, the production of peptide and steroid hormones, growth factors, growth factor binding protein, prostaglandins, cyclic nucleotides, protein kinases and transcription factors by cells isolated from bovine, porcine, rabbit, chicken and human ovarian and oviduct cells, oocyte-cumulus complexes and embryos can be an efficient means of studying the effects, mechanisms of action and safety of various biologically active substances.

The steroidogenic acute regulatory (StAR) protein plays essential roles in the delivery of cytosolic cholesterol into the mitochondrial inner membrane, which is an acute regulated and rate-limiting step for the steroid hormone synthesis. Since retinoic acids (RAs) are known to induce the synthesis of steroid hormones in mouse Leydig cells in vitro, mouse Leydig tumour cells, K28, were used to determine the effect of RAs on the level of StAR mRNA by Northern blot analysis. The level of StAR mRNA reached the maximum in a 4-8 h treatment with all-trans-RA (atRA) or 9-cis-RA (9cRA), and the effects were dose-dependent. The effect of 9cRA on the levels of StAR mRNA was blocked by actinomycin D, which indicates that 9cRA might exert a direct effect on the transcription of the gene. Promoter/reporter constructs containing a 5'-flanking region of the mouse or rat StAR gene were prepared, and luciferase activity was assayed following transient transfection into K28 or adrenal tumour cells, Y1. The result revealed that the luciferase activity was increased by 4-5-fold in response to the treatment of 9cRA, which indicated that 9cRA participates transcriptional activation of the StAR protein gene.

To identify a new gene(s) located on the yeast artificial chromosome (YAC) clone D142H8 that was mapped to human chromosome 21q22.1, purified YAC DNA from the clone was utilized directly as a probe to screen a human brain cDNA library after the suppression of human repetitive DNA. One cDNA clone hybridizing specifically to the YAC D142H8 DNA was identified. The clone has an insert of 1341 bp and the longest open reading frame of 349 amino acids. A search of GenBank revealed that the clone has a high degree of homology to zeta-crystallin (quinone reductase) at the amino acid level, and its nucleotide sequence represents the expressed sequence from the 50-kb segment of the human chromosome 21q11.1. Thus a new gene was named CRYZL1 (zeta-crystalline-like 1). Genomic Southern blot with total human and yeast DNAs suggests that CRYZL1 might be a single-copy gene. The fluorescence in situ hybridization procedure was applied, and the results showed that the gene mapped to the human chromosome 21q22.1 subband. The CRYZL1 mRNA was expressed in heart, brain, skeletal muscle, kidney, pancreas, liver, and lungs but at different levels in different tissues.

Studies were carried out to define the hormone-induced signal transduction pathway during maturation of Rana dybowskii oocytes. Rapamycin, a specific inhibitor of S6 kinase, blocked progesterone-induced oocyte germinal vesicle breakdown (GVBD) in a dose-dependent manner indicating that S6 kinase is required for meiotic maturation of Rana oocytes. Addition of rapamycin within 3 h, but not 6 h, of progesterone treatment inhibited GVBD. In contrast, cycloheximide, a general protein synthesis inhibitor, blocked GVBD even when added 9 h after progesterone addition. A twofold increase in S6 kinase activity occurred within 1 h of progesterone stimulation and rapamycin inhibited this activity. Rapamycin also suppressed, in a dose-dependent manner, progesterone-induced protein synthesis during the first 12 h of culture but less effectively later. Histone H1 kinase activity (maturation-promoting factor, MPF) was observed in oocyte extracts at two different times (between 6 and 9 h and at 24 h) following progesterone stimulation. Rapamycin blocked H1 kinase activity between 6 and 9 h of culture but not that observed at 24 h. In contrast, cycloheximide suppressed progesterone-induced H1 kinase activity as well as protein synthesis throughout the course of incubation. Such results indicate that rapamycin and cycloheximide have common and unique effects on oocyte maturation and suggest that progesterone-induced S6 kinase activity is closely associated with induction of protein synthesis and activation of MPF during oocyte maturation. Results in Rana contrast with those obtained in Xenopus where rapamycin inhibited S6 kinase but failed to inhibit GVBD or protein synthesis. Differences in the response of Rana and Xenopus oocytes to rapamycin are discussed in relation to seasonal, biochemical, and species variations.

Pituitary adenylate cyclase-activating polypeptide (PACAP), a novel neuropeptide with considerable homology to vasoactive intestinal peptide and GH-releasing hormone, exists in two biologically active forms, PACAP-38 and -27. The presence of PACAP in the ovary has been demonstrated, where it stimulates steroidogenesis and cAMP accumulation in cultured granulosa cells. In the present study, gonadotropin regulation of PACAP gene expression was examined in PMSG/human (h)CG-treated immature rat ovaries and cultured preovulatory follicles. Northern blot analysis of ovaries obtained from PMSG/hCG-treated immature animals revealed the transient induction of PACAP transcripts by hCG, reaching a maximum at 6 h. The major cell types expressing PACAP messenger RNA were granulosa cells of preovulatory follicles and some theca/interstitial cells. In preovulatory follicles cultured in serum-free medium, PACAP transcripts were transiently induced by LH and FSH, reaching a maximum 6-9 h after stimulation in granulosa cells but not in theca cells. Treatment with cycloheximide or alpha-amanitin abolished LH-induced PACAP transcripts, indicating that new protein synthesis and transcription are necessary. Treatment with MDL-12,330A, an inhibitor of adenylate cyclase, inhibited LH-induced PACAP messenger RNA, and forskolin mimicked the LH action, implying the role of adenylate cyclase activation. In contrast, treatment with chelerythrine, an inhibitor of protein kinase C, and 2-O-tetradecanol-phorbol-13-acetate had no effect. We further tested the role of PACAP in follicle apoptosis using apoptotic DNA fragmentation analysis. Treatment with PACAP-38 suppressed follicle apoptosis in a dose-dependent manner. Moreover, the LH suppression of follicle apoptosis was partially blocked by cotreatment with PACAP-38 antagonist, indicating mediation by endogenous PACAP-38. These results suggest that PACAP, transiently induced by the gonadotropin surge, could be a local regulator of a number of events and may act as a follicle survival factor during the periovulatory period.

The aim of our in vitro experiments with isolated porcine ovarian follicles was to study the effects of gonadotropins, GH, IGF-I and oxytocin (OT) on release of ovarian steroid, OT, IGF-I, insulin-like growth factor-binding protein-3 (IGFBP-3), prostaglandin F (PGF), prostaglandin E (PGE) and cAMP. It was found that quarters of ovarian follicles cultured for 8 days produced significant amounts of progesterone, estradiol-17 beta, OT and IGFBP-3 with peaks of accumulation from the 3rd to the 8th day of culture. Addition of serum promoted progesterone, estradiol and OT release, whilst accumulation of IGFBP-3 was maintained to a greater extent in serum-free medium. GH (10 ng/ml or above) was able to inhibit androstenedione, OT, PGF and IGFBP-3, to stimulate IGF-I and cAMP, and to alter testosterone and PGE release by follicles cultured in serum-supplemented and/or serum-free medium. IGF-I (10 ng/ml or more) inhibited androstenedione and PGF secretion, stimulated testosterone, estradiol, OT and cAMP production, but did not influence progesterone, IGFBP-3 or PGE output in these conditions. OT (100 ng/ml) was able to inhibit androstenedione and to stimulate testosterone, IGF-I, PGF and PGE, but not estradiol or IGFBP-3 release. A stimulatory effect of LH on progesterone and OT and an inhibitory influence of LH on estradiol secretion in the serum-supplemented medium were observed. FSH in these conditions stimulated OT, but not progesterone or estradiol secretion. The use of this experimental model suggests the involvement of gonadotropins, OT, GH and IGF-I in the control of ovarian steroid and nonapeptide hormone, growth factor, growth factor-binding protein, prostaglandin and cyclic nucleotide production. The stimulatory effect of GH on IGF-I, and the stimulatory influence of IGF-I on OT, as well as coincidence of the majority of effects of IGF-I and OT, suggest the existence of a GH-IGF-I-OT axis. On the other hand, the different patterns of action of GH and IGF-I on OT, estrogen and IGFBP-3 suggest that part of the GH effect on ovarian cells is IGF-I independent.

Seasonal changes in testicular structure and function in three species of Korean frogs ( Rana nigromaculata, R. rugosa, and R. dybowskii ) having different breeding seasons and habitats were examined throughout the year. R. nigromaculata live in rice fields and breed in early May. Their gonadosomatic index (GSI) and testis weight decreased slightly from May until July, but increased markedly from August to high levels through December. The diameter (cross-sectional area) of seminiferous tubules changed little from January until August, increased sharply from September through October, and decreased thereafter. In seminiferous tubules, the number of primary spermatogonia (I SPG) was low from January–March, increased from April to maximum levels by May–June, and decreased subsequently. The number of spermatids (SPT) was highest from November to March, decreased to nondetectable levels in May, and increased markedly from September to November. Spermiation was most active during March and April. R. rugosa, which live in streams and breed during May–June, exhibited no changes in GSI, testis weight, or seminiferous tubule size throughout the year. The number of I SPG was high during May–August and that of secondary spermatogonia (II SPG) was highest in August. The number of SPT increased to high levels in November–December. Active spermiation occurred from January to April in this frog. In R. dybowskii, which live in the mountains and breed from late February to early March, the number of I SPG gradually increased from April through August; however, essentially no other spermatogenic cells were observed from January to July. A marked increase in early spermatogenic cells appeared during August–September and was followed by an increase in SPT from November to December. From December to March the number of spermatozoa increased and spermiation occurred. In general, testicular testosterone levels were high in the winter and low in summer in all three species, and positively correlated with the number of interstitial cells and the size of their nucleus.

The present study is designed to evaluate the relationship between gonadotropin-releasing hormone (GnRH) and GnRH receptor (GnRHR) gene expression during the steroid-induced LH surge. One week after ovariectomy (OVX), a capsule containing 17beta-estradiol (E) or vehicle (V) was implanted into OVX rats, and 2 days later a single injection of progesterone (P) or V was administered s.c. at 10:00 h. Poly(A)-rich RNA samples were isolated from the micropunches of the preoptic area (POA) and the posterior mediobasal hypothalamus (pMBH) from both sides of individual brain slices. Using competitive reverse transcription-polymerase chain reaction (RT-PCR) procedures, three parameters (POA GnRH, pMBH GnRHR) and pituitary GnRHR mRNA levels were simultaneously determined in each individual animal. POA GnRH mRNA and pituitary GnRHR mRNA levels were decreased by treatment with E, but increased by a combination of E and P. In contrast, pMBH GnRHR mRNA levels were clearly augmented by treatment with E, and decreased by the combination of E and P. Temporal changes in such parameters were determined in OVX+E+V- and OVX+E+P-treated rats. P augmented POA GnRH mRNA levels at the time of the LH surge (17:00 h) and the increased GnRH mRNA levels were remained until 22:00 h, while E alone failed to alter POA GnRH mRNA levels. In the pMBH micropunch samples, P substantially decreased E-induced increase in GnRHR mRNA levels at 17:00 h and further lowered those until 22:00 h. Antisense oligonucleotides of GnRHR mRNA administered into the lateral ventricle of OVX+E-treated rats blocked the E-induced increase in pMBH GnRHR mRNA levels. The antisense oligonucleotides also prevented the LH surge as well as the increase in pituitary GnRHR mRNA levels in the OVX+E+P-treated group. However, administration of this antisense oligonucleotides failed to alter POA GnRH mRNA levels. In conclusion, the present study demonstrated that there is an inverse relationship between POA GnRH mRNA levels and pMBH GnRHR mRNA levels in response to E and/or P, and that the blockade of the E-induced increase in pMBH GnRHR mRNA levels effectively nullified the P-induced LH surge. These results indicate that pMBH GnRHR gene expression is involved in synchronizing the GnRH neuronal activity, which is crucial for the generation of the LH surge.

To examine whether an ultrashort feedback mechanism of gonadotropin-releasing hormone (GnRH) operates at the level of gene transcription, we studied the effects of GnRH analogs on GnRH promoter activity and GnRH mRNA level in hypothalamic GT1–1 neuronal cells. Treatment of GT1–1 cells with buserelin, a GnRH agonist, or native GnRH for 24 h significantly decreased GnRH promoter activity and its mRNA level, whereas that with GnRH antagonists, antide or [ D -Phe 2 , D -Ala 6 ]-GnRH, showed no effect. The inhibitory effects of buserelin on GnRH gene transcription and GnRH mRNA level were dose-related, and a significant inhibition was observed in cells treated with buserelin at concentrations higher than 0.1 μM. Time-course experiments showed that significant decreases in GnRH promoter-driven luciferase activity and GnRH mRNA level were observed within 12 h and sustained up to 48 h. Moreover, treatment with GnRH agonist for 12 h significantly decreased the transcription rate of the mouse GnRH gene, as revealed by nuclear run-on transcription assay. The promoter analysis with the 5′-deletional constructs demonstrated that cis -acting elements important for GnRH autoregulation by GnRH agonist reside within −854 bp upstream from the transcription start site. These data clearly demonstrate that GnRH can exert autocrine regulation at the level of GnRH gene transcription.

Experiments were carried out at different times of hibernation to ascertain whether prostaglandin is produced by Rana ovarian follicles during gonadotropin- or 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced in vitro ovulation. Ovarian fragments were cultured in amphibian Ringer in the presence or absence of frog pituitary homogenate (FPH, 0.05 gland/ml) or TPA (1 or 10 microM). After various periods of culture, incidence of ovulation was determined and prostaglandin F2 alpha (PGF2 alpha) accumulated in culture medium was measured by radioimmunoassay. FPH and TPA increased PGF2 alpha levels in medium in a dose-dependent manner. The time course of PGF2 alpha secretion and ovulation by FPH or TPA treatment varied during the hibernation period. In early-hibernation, FPH stimulated neither PGF2 alpha secretion nor ovulation while TPA stimulated PGF2 alpha secretion, although it failed to induce ovulation. In mid-hibernation, FPH and TPA effectively stimulated PGF2 alpha secretion and ovulation, but both events took place several hours later than those observed in late-hibernation. Some fragments obtained in mid-hibernation and most obtained in late-hibernation spontaneously produced PGF2 alpha in vitro without FPH or TPA treatment and in some instances spontaneously ovulated. Furthermore, FPH or TPA increased PGF2 alpha levels further or accelerated the time course of secretion by such fragments. In late-hibernation, PGF2 alpha secretion induced with FPH or TPA increased simultaneously with or later than onset of ovulation. Exogenous cAMP (2.5 mM) or 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7, 100 microM), a PKC inactivator, markedly suppressed FPH- or TPA-stimulated PGF2 alpha secretion and ovulation in mid-hibernation. Indomethacin (IM, 5 micrograms/ml) strongly suppressed TPA- or FPH-stimulated PGF2 alpha production by fragments but its effect on ovulation varied among animals and at different times. IM suppressed ovulation of some ovarian fragments obtained in mid-hibernation, but failed to suppress hormone-induced ovulation in late-hibernation. Cycloheximide (5 micrograms/ml) and actinomycin D (1 microgram/ml) effectively suppressed FPH-stimulated PGF2 alpha production and ovulation, whereas actinomycin D reduced but failed to significantly suppress TPA-induced PGF2 alpha production in mid-hibernation. In general, effects of ovulation inhibitors exhibited strong seasonal variations and were less efficient as the breeding season approached. Taken together, the data suggest that elevated levels of PGF2 alpha are associated with spontaneous and hormone-induced ovulation, and PKC mediates gonadotropin induction of PGF2 alpha but not steroid synthesis in Rana ovaries.

Using frog ovarian follicles in vitro we assessed the role of the theca/epithelium (THEP) layer and granulosa cells in the production of steroids. The THEP layer and granulosa cell-enclosed oocytes (GCEOs) were separated from ovarian follicles of the frog, Rana nigromaculata, by microdissection. Intact follicles (IFs), THEP layer, or GCEOs were cultured for 6 hr in amphibian Ringer's in the presence or absence of various steroid precursors (100 ng/ml) or of frog pituitary homogenate (FPH, 0.05 gland/ml). The amounts of progesterone (P4), 17 alpha-hydroxyprogesterone (17 alpha-OHP4), androstenedione (AD), testosterone (T), and estradiol (E2) secreted into the medium by the different types of follicular components during culture were measured by radioimmunoassay. Addition of pregnenolone resulted in a marked increase in P4 concentrations produced by GCEOs (1858 pg/follicle) and IFs (1549 pg/follicle), but a much smaller increase in P4 occurred in the THEP layer (427 pg/follicle). Likewise, addition of P4 stimulated greater production of 17 alpha-OHP4 by GCEOs (659 pg/follicle) and by IFs (877 pg/follicle) than by the THEP layer (330 pg/follicle). Exogenous 17 alpha-OHP4 markedly increased AD levels secreted by GCEOs (1139 pg/follicle) and by IFs (841 pg/follicle), but not by the THEP layer (< 15 pg/follicle). In contrast, AD addition resulted in the secretion of markedly elevated levels of T by the THEP layer (2326 pg/follicle) and IFs (3652 pg/follicle) as compared to the GCEOs (408 pg/follicle). Addition of T markedly increased E2 concentrations produced by GCEOs (378 pg/follicle) and by IFs (673 pg/follicle), but not by the THEP layer (< 12 pg/follicle).(ABSTRACT TRUNCATED AT 250 WORDS)

Previous studies have demonstrated that direct or indirect elevation of cAMP levels in cultured amphibian ovarian follicles simultaneously stimulated production of oocyte maturation-inducing steroid (progesterone) by the follicles and inhibited oocyte maturation induced by endogenous or exogenous hormone. The duration of cAMP stimulation influenced arrest and reinitiation of oocyte meiotic maturation in ovarian follicles of Rana dybowskii. Addition of forskolin (adenylate cyclase stimulator) to cultured follicles inhibited both progesterone- and frog pituitary homogenate (FPH)-induced oocyte maturation. Similar inhibitory results were obtained when hormone-treated follicles were cultured in the continual presence of cAMP. Oocyte maturation increasingly occurred in follicular oocytes when cAMP or forskolin addition was delayed following treatment with FPH or progesterone. Transient exposure (6-8 hr) of ovarian follicles to forskolin or cAMP markedly stimulated oocyte maturation as well as accumulation of progesterone as measured by radioimmunoassay within the ovarian follicles. Forskolin was more effective than cAMP, at the dose tested, in stimulating progesterone production and accumulation by the follicles. The data demonstrate that transient manipulation (elevation) of cAMP levels in cultured follicles, without added FPH or steroid, was sufficient to initiate oocyte maturation. Results suggest that, with transient exposure to forskolin or exogenous cAMP, there is a sequential increase and decrease in endogenous cAMP levels in the somatic cells and germ cell components of the ovarian follicle. These changes appear to mediate production of maturation-inducing steroid and secondarily allow its effects on the oocyte to be expressed.

Seasonal and hormonal influences regulating oocyte maturation (germinal vesicle breakdown, GVBD) in ovarian follicles of Rana dybowskii were investigated. During the early winter (Dec.-Jan.) GVBD occurred at a low incidence following in vitro culture of intact follicles. Addition of progesterone of frog pituitary homogenate (FPH) to such follicles induced oocyte maturation, whereas IBMX or forskolin inhibited hormone-induced oocyte maturation. The time course of spontaneous in vitro maturation varied markedly with the seasons and between animals. Follicles isolated from the ovaries in early February required 21-24 hours of culture to mature spontaneously, and addition of FPH or progesterone to the culture medium markedly accelerated the time course of GVBD. In contrast, follicles isolated in late February matured very rapidly (less than 6 hours), and FPH or progesterone were ineffective in accelerating the time course of GVBD. IBMX and forskolin separately or in combination stimulated follicular progesterone production, which resembled that seen following FPH stimulation. FPH addition to such follicles shifted the steroid peak to the left (accelerated) and increased the absolute amount of hormone detected in late-maturing follicles (50% GVBD, about 18 hours) but not in rapidly maturing follicles (50% GVBD, 3 hours). In contrast to other amphibians, a high incidence of spontaneous oocyte maturation occurred during in vitro culture. Essentially all animals exhibited spontaneous maturation during the normal breeding season, even those animals collected in the early winter and kept in artificial hibernation at 4 degrees C for extended periods.