Receptors and secretory actions of sigma/phencyclidine agonists in anterior pituitary cells

Opiate receptor subtypes in the adenohypophysis were analyzed by binding studies with tritiated etorphine, phencyclidine (PCP), and N-allylnormetazocine [(+)SKF 10,047] in anterior pituitary cell (AC) cultures and membranes, and in cell populations separated by centrifugal elutriation. In cultured AC, specific binding of [3H]etorphine revealed two sets of saturable sites with Kd values of 5 nM and about 10 microM. The high affinity [3H]etorphine sites were present in low concentration and represent specific opiate receptors that mediate the direct inhibitory actions of etorphine and morphine on LH release in vitro. The more abundant low affinity sites, observed in the presence of higher concentrations of unlabeled opiates, exhibited the properties of sigma/PCP receptors. In intact AC and pituitary membranes, specific [3H]PCP binding was saturable with respect to labeled and unlabeled ligand concentrations, and Scatchard analysis revealed a single class of relatively high affinity [3H]PCP-binding sites (Kd = 98 nM in pituitary membranes). Relative potencies derived from inhibition of [3H]PCP binding in AC by PCP-related drugs were: (-) cyclazocine greater than dexoxadrol greater than N-[1-(2-Thienyl)cyclohexil]piperidine greater than PCP greater than (+)SKF 10,047 greater than levaxodral greater than (+)cyclazocine less than (-)SKF 10,047 greater than (+)ethylketocyclazocine greater than haloperidol greater than (-)ethylketocyclazocine. In elutriated pituitary cells, specific [3H]PCP binding was correlated with the LH content of the individual cell fractions. The binding of (+)-[3H]SKF 10,047 was also specific and saturable in AC and anterior pituitary membranes, which contained two classes of binding sites with Kd values of 87 nM and 3.3 microM. In fractionated pituitary cells, specific binding of (+)-[3H]SKF 10,047 was similar in enriched lactotrophs and gonadotrophs. The high affinity class of (+)-[3H]SKF 10,047-binding sites probably corresponds to sigma-receptors, and the low affinity class to PCP receptors. In contrast to the inhibitory actions of opiates on LH release in vitro, PCP and (+)SKF 10,047 stimulated LH release in cultured AC and enhanced the secretory responses to GnRH as well as KCl. The stimulation of LH release by PCP was dependent on extracellular calcium and is probably related to increased transmembrane calcium influx. The stimulatory sites may correspond to selective sigma/PCP receptors, and could represent a distinct nonopiate receptor subtype with the potential for modulation of gonadotropin secretion.     

Peripheral-type benzodiazepine receptors in endocrine organs: autoradiographic localization in rat pituitary, adrenal, and testis

We have used [3H]Ro5-4864, a ligand selective for peripheral-type benzodiazepine receptors, to identify and localize peripheral-type benzodiazepine receptors in endocrine organs. Autoradiographic studies reveal an uniform distribution of [3H]Ro5-4864 binding sites within the anterior, intermediate, and posterior lobes of the pituitary gland, with highest concentrations present in the posterior pituitary. In rat adrenal gland, specific binding sites for [3H]Ro5-4864 are found only in the adrenal cortex, with highest density in the zona glomerulosa and significantly lower concentrations in the zona fasciculata and zona reticularis. [3H]Ro5-4864-associated silver grains in the testis are intensely localized over the interstitial tissue; low concentrations of silver grains are present over the epithelium of the seminiferous tubules but are absent from the tubular lumen. These studies demonstrate a differential and discrete localization of peripheral-type benzodiazepine receptors in rat pituitary, adrenal, and testis.     

The presence of corticotropin-like and opiate-like activities in tissues of adult sea lamprey, Petromyzon marinus L

Sexually mature landlocked sea lamprey were captured during their upstream migration. Different tissues, including the brain, pituitary, heart, liver, gut, testis, and ovary, were dissected from the animals and an acetone powder was prepared from each tissue. The tissue acetone powders were subjected to heat treatment and were then extracted with an acidic medium in order to inactivate any proteases present. The resulting acid acetone powders were then tested for their ability to stimulate corticosterone production from isolated rat adrenal cells and to displace the binding of D-Ala2-D-Leu5-[tyrosyl-3,5-3H]-enkephalin to rat brain membranes. It was found that the brain and liver contained steroidogenic activity while opiate activity was detected in the heart, liver, gut, brain, and pituitary. No steroidogenic activity was found in the heart, ovary, testis, gut, and pituitary while ovary and testis did not contain assayable opiate activity. None of the tissues contained beta-endorphin-like immunoreactivity.     

Angiotensin-converting enzyme localized in the rat pituitary and adrenal glands by [3H]captopril autoradiography

We have localized angiotensin-converting enzyme (EC 3.14.5.1) in the rat pituitary and adrenal glands by [3H]captopril autoradiography. The maximal numbers of [3H] captopril-binding sites are: posterior pituitary, 4500 fmol/mg protein; anterior pituitary, 1950 fmol/mg; intermediate pituitary, less than 100 fmol/mg; adrenal medulla, 480 fmol/mg; and adrenal cortex, less than 25 fmol/mg. The distribution within the posterior pituitary and adrenal medulla is homogeneous, whereas that in the anterior pituitary is patchy. Subcellular fractionation of the bovine adrenal medulla reveals enrichment of angiotension-converting enzyme in plasma membrane fractions, but not in chromaffin granules. [3H]Captopril autoradiography in the rat pituitary gland is unaltered by dehydration, adrenalectomy, or reserpine treatment and in Brattleboro rats. [3H]Captopril binding in the adrenal medulla is increased by 75% 3 weeks after hypophysectomy and is elevated by 80% after reserpine treatment. The change after hypophysectomy is not reversed by dexamethasone treatment.     

Enkephalin convertase demonstrated in the pituitary and adrenal gland by [3H]guanidinoethylmercaptosuccinic acid autoradiography: dehydration decreases neurohypophyseal levels

[3H]Guanidinoethylmercaptosuccinic acid (GEMSA) autoradiography demonstrates the particulate form of a carboxypeptidase B-like peptide processing enzyme, enkephalin convertase (EC 3.4.17.10), in the rat pituitary and adrenal glands. The maximal number of binding sites (Bmax) for [3H]GEMSA is 20 pmol/mg protein in the intermediate lobe of the pituitary, 12.0 pmol/mg protein in the posterior pituitary lobe, 15 pmol/mg protein in the anterior pituitary lobe, 5.8 pmol/mg protein in the adrenal medulla, and less than 0.3 pmol/mg protein in the adrenal cortex. The labeling pattern is homogeneous within each of these regions. Subcellular fractionation of the bovine adrenal medulla demonstrates that [3H] GEMSA-binding sites are localized to chromaffin granules. In Brattleboro rats and dehydrated rats, the level of posterior pituitary [3H]GEMSA binding is less than 25% of that in control animals. This decrease is abolished by arginine vasopressin treatment of Brattleboro rats or rehydration of dehydrated rats. There are no changes in [3H] GEMSA binding in the supraoptic nucleus or magnocellular portion of the paraventricular nucleus of the hypothalamus under any of these conditions, suggesting that the alterations observed in the neurohypophysis result from an increased rate of loss of enkephalin convertase. The level of anterior pituitary enkephalin convertase is unchanged by dehydration, adrenalectomy, or dexamethasone or in Brattleboro rats. [3H]GEMSA labeling in the intermediate pituitary lobe is unaffected by dehydration and haloperidol treatment and in Brattleboro rats. The adrenal medullary enzyme is not altered by reserpine, hypophysectomy, or splanchnic denervation or in Brattleboro rats.     

Localization and secretion of inhibin/activin subunits in the human and subhuman primate fetal gonads

Little is known about the ability of the fetal primate gonads to produce inhibin/activin. We investigated the presence of the alpha-, beta A-, and beta B-subunits of inhibin/activin in fetal human (16-23 weeks gestational age) and rhesus monkey (days 150-157 of gestation; term = 165 days) testes and ovaries by immunocytochemistry. The regulation of alpha-inhibin secretion by gonadotropins was studied in fetal testicular cultures. In the human fetal testis, alpha-subunit immunostaining was found in interstitial and intratubular cells, while beta A- and beta B-subunit immunostaining occurred in clusters of Leydig cells that were clearly demarcated from groups of Leydig cells that were immunonegative. In the late gestational monkey testis, the alpha-subunit was localized in tubular cells, and the beta B-subunit was present in the tubules and interstitium. Testicular cells from midgestation human testes secreted detectable immunoreactive alpha-inhibin in response to FSH and hCG stimulation; alpha-inhibin levels were significantly higher after hCG than FSH. In contrast, levels of alpha-inhibin secreted by rhesus monkey testicular cells were significantly increased by FSH, but not hCG. In the ovary, only weak beta B-subunit immunoreactivity was detected in granulosa cells of a few primary follicles from midgestational human fetal ovaries. In contrast, all three subunits were found in granulosa cells of numerous primary and secondary follicles in the late gestation rhesus monkey ovary. In light of recent evidence that inhibins/activins have actions on gonadal differentiation and growth modulation in vitro, as well as endocrine effects on the fetal pituitary, we propose that these proteins may have intragonadal and endocrine roles in human and subhuman intrauterine gonadal development.     

Immunohistochemical analysis of cytochrome P-450 17 alpha-hydroxylase in pig adrenal cortex, testis and ovary

Cytochrome P-450 specific for steroid 17 alpha-hydroxylation (P-450(17 alpha] was immunohistochemically observed in pig adrenal cortex, testis and ovary by the biotin-streptavidin method using a specific antibody against P-450(17 alpha) purified from neonatal pig testis. In the adrenal cortex, P-450(17 alpha) was present in the zona fasciculata and reticularis while no immunoreactivity was observed in the zona glomerulosa, confirming the absence of 17-hydroxylated steroid synthesis in the zona glomerulosa. In the testis, P-450(17 alpha) was present exclusively in Leydig cells and immunoreactivity was absent in seminiferous tubules. In the ovary, immunoreactivity was observed only in the theca interna but not in the membrana granulosa of follicles. Among the tissues examined, the relative intensity of immunoreactivity was greatest in the Leydig cells, and progressively less in theca interna cells, outer fasciculata cells and inner fasciculata and reticularis cells.     

Serotonin and dopamine receptors in the rat pituitary gland: autoradiographic identification, characterization, and localization

S2 serotonin and D2 dopamine receptors were identified, characterized, and localized in rat pituitary gland by quantitative light microscopic autoradiography. [3H]Spiperone was used to localize S2 serotonin and D2 dopamine receptors. A high concentration of D2 dopamine receptors [1 microM 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene (ADTN)- or sulpiride-displaceable [3H]spiperone binding] was found in the rat intermediate lobe with much lower concentrations present in the anterior and posterior lobes. Significant densities of cinanserin-displaceable [3H]spiperone binding sites (i.e. S2 serotonin receptors) were present in all three lobes of the pituitary gland. [125I]Lysergic acid ([125I]LSD) was used to characterize further and selectively visualize S2 serotonin receptors in the rat pituitary. Data analysis by densitometry showed that [125I]LSD binding the rat intermediate pituitary was saturable and of high affinity with an apparent dissociation constant (Kd) of 1.2 nM. Data from competition studies using a variety of compounds showed a S2 serotonin receptor profile at this [125I]LSD binding site in rat pituitary. The highest concentration of [125I]LSD binding sites was found in the intermediate lobe with progressively lower concentrations present in the posterior and anterior lobes, respectively. There is a uniform pattern of distribution of S2 serotonin and D2 dopamine receptors within each lobe of the rat pituitary gland. The results of the present study provide the first identification of S2 serotonin receptors in the pituitary and confirm the heterogeneous distribution of D2 dopamine receptors within the rat pituitary. These data provide further evidence for the importance of dopamine in regulating pituitary function and suggest a physiological role for serotonin in regulating pituitary hormone secretion.     

Phencyclidine in nanomolar concentrations binds to synaptosomes and blocks certain potassium channels.

Phencyclidine [1-(phenylcyclohexyl)piperidine; PCP], in low dose (approximately equal to 0.1-0.2 mg/kg of body weight), induces a schizophrenia-like behavioral syndrome in man; this effect has been attributed to block of neuronal K channels. We used a K-stimulated 86Rb efflux assay to demonstrate that low concentrations of PCP (10-50 nM) block a class of depolarization-activated K channels in rat brain synaptosomes--pinched-off presynaptic nerve terminals. The dose-response curve is biphasic, and much higher PCP concentrations (greater than 10 microM) are required to block the remainder of the K-stimulated 86Rb efflux. The [3H]PCP binding curve for synaptosomes is also biphasic: PCP binds to some components with high affinity (Kd approximately equal to 6.0 X 10(-8) M), and to other components with much lower affinity (Kd approximately equal to 1.15 X 10(4) M). PCP can be photoactivated with UV light to form covalent bonds: after UV irradiation, previously-bound [3H]PCP is no longer displaceable by a large excess of unlabeled PCP. Preliminary data from NaDodSO4/polyacrylamide gel electrophoresis studies after covalent binding of [3H]PCP to synaptosomes, suggest that the high-affinity binding site may be on a large protein (Mr approximately equal to 220,000). We conclude that the high-affinity PCP binding protein is associated with the K channels that are blocked by nanomolar concentrations of PCP. Block of these channels could, by prolonging action-potential duration in presynaptic nerve terminals, enhance calcium entry and neurotransmitter release, thereby altering transmission at central synapses involved in behavioral expression.     

Autoradiographic localization of peripheral benzodiazepine, dihydroalprenolol and arginine vasopressin binding sites in the pituitaries of control, stalk transected and Brattleboro rats

The autoradiographic distribution of [3H]arginine vasopressin, [3H]spiperone, [3H]GABA, [3H]dihydroalprenolol and the peripheral-type benzodiazepine ligand [3H]Ro5-4864 were examined in the rat pituitary before and after pituitary stalk transection. Stalk transection produced dramatic changes in the cellular architecture of the pars nervosa. Glial fibrillary acidic protein, an astrocyte marker reported in pituicytes, increased after stalk transection, whereas neurofilament, a marker for neuronal innervation, was lost. These structural changes demonstrated a successful stalk transection, permitting interpretation of changes in the densities of several [3H]-ligands over the three lobes. [3H]Ro5-4864 binding was markedly increased, suggesting that this site was located on the pituicytes. Conversely [3H]spiperone and [3H]arginine vasopressin binding density over the pars nervosa decreased. In the mutant diabetes insipidus rat (Brattleboro), which lacks pituitary vasopressin, [3H]arginine vasopressin binding was undetectable in the pars nervosa. [3H]dihydroalprenolol and [3H]GABA binding sites were unchanged by the lesion. These results are discussed in terms of the occurrence of functional acceptors on pituicytes and their possible role in neurohydrophyseal secretions.     

Sexual differences of steroidogenic enzymes in embryonic gonads of the chicken (Gallus domesticus)

The left ovary and testis of 15-day-old embryos of the chicken were compared in the enzyme activities related to steroidogenesis. The activity of delta 5-3 beta-hydroxysteroid dehydrogenase coupled with delta 5-delta 4 isomerase in the ovary was similar to that of the testis. Activities of 17 alpha-hydroxylase and C-17-C-20 lyase in the ovary were 2.5 and 2.6 times those in the testis. From the CO-induced difference spectrum, the content of cytochrome P-450 in the ovarian microsomes was estimated as 27.6 pmol/mg protein. However, no detectable amount of cytochrome P-450 was observed in the testicular microsomal fraction. The substrate (progesterone)-induced difference spectrum was appreciable only in the ovarian microsomes. The activity of microsomal NADPH-cytochrome c reductase in the ovary was significantly higher than that in the testis. The activities of 17 beta-hydroxysteroid dehydrogenase in both gonads were similar to each other, when androstenedione was used as substrate. However, its activity in the ovary was 1.4 and 3.1 times that in the testis, when dehydroepiandrosterone and estrone were used as substrate, respectively. Aromatase activity in the ovary was over 100 times that in the testis, as assessed by release of [3H]water from [1-3H]testosterone. Appreciable amounts of radioactive estradiol-17 beta and estrone were formed from [4-14C]testosterone and [7-3H]androstenedione, respectively, only by the ovarian tissue. 5 beta-Reductase activity in the ovary was 1.4 times that in the testis.     

Biosynthesis of 15-hydroxylated steroids by gonads of the river lamprey, Lampetra fluviatilis,in vitro

Ovaries and testes of the river lamprey, Lampetra fluviatilis, were incubated with [³H]testosterone and [³H]progesterone and the major metabolites identified by chromatography, chemical reaction, and gas chromatography-mass spectrometry. The major metabolite of progesterone with gonads of both sexes was 15α-hydroxyprogesterone (26.6% yield in ovary, 50.0% in testis); a small amount of 15β-hydroxyprogesterone was also found in the testis incubation. With testosterone as substrate, the major metabolite was 15β-hydroxytesterone (84.8% in testis, 65.2% in ovary). The formation of androstenedione, but not of testosterone, from progesterone was also demonstrated in both sexes.     

Identification and characterization of arginine vasopressin receptors in the rat testis

We have previously shown that arginine vasopressin (AVP) directly inhibits testicular steroidogenesis in vitro. In the present study, binding of neurohypophysial peptides to interstitial cells of the rat testis was studied using [3H]AVP as the ligand. Interstitial cells were obtained from adult rat testis after collagenase dispersion and were incubated with [3H]AVP in the presence or absence of unlabeled AVP. Binding equilibrium was reached by 60 min at 4 C, while incubation at higher temperatures (23 and 37 C) resulted in an apparent decrease in binding. Scatchard plot analysis of equilibrium binding data revealed the existence of one class of high affinity, low capacity binding sites (Kd = 1.0 +/- 0.3 nM; maximal binding = 8.5 fmol/10(6) cells). In addition, the rate constants of association and dissociation were calculated to be 0.024 nM-1 min-1 and 0.009 min-1, respectively. Addition of naturally occurring neurohypophysial hormones as well as their synthetic analogs inhibited [3H]AVP binding to testis cells, resulting in parallel displacement curves. The order of potencies for the native peptides was: AVP = lysine vasopressin = arginine vasotocin (IC50, 5 X 10(-10) M) greater than oxytocin = mesotocin (IC50, 4 X 10(-7) M) greater than isotocin = glumitocin (IC50 greater than 10(-6) M). Furthermore, two potent vasopressor antagonists, d(CH2)5Tyr(Me)AVP ([1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid), 2-(O-methyl)tyrosine]AVP) and dPTyr(Me)AVP ([1-deaminopenicillamine-2-(O-methyl)tyrosine]AVP) competed for [3H]AVP binding with a higher affinity (IC50, approximately 10(-11) M) than native AVP. In contrast, a selective antidiuretic agonist, dDAVP (1-deamino-8-D-AVP), only competed weakly for receptor binding, while a specific oxytocic agonist, (Thr4,Gly7)oxytocin, did not affect AVP binding. These results suggested that the testis may contain the V1 receptor subtype. Studies on the intratesticular distribution of AVP receptors indicated minimal binding to cells derived from the seminiferous tubule, while most of the AVP-binding sites sediment with enriched fractions of Leydig cells after Metrizamide density gradient centrifugation. AVP-binding sites were also found in rat liver, kidney, and anterior pituitary (10.7, 2.6, and 1.7 fmol/mg protein), whereas adrenal, cerebellum, prostate, and hypothalamus were devoid of AVP-binding sites. Thus, we have demonstrated the presence of high affinity, stereospecific receptors for AVP in the interstitial cell compartment of the rat testis. These V1 receptors may mediate the direct inhibitory action of neurohypophysial hormones on testicular Leydig cell steroidogenesis.     

In vitro effects of follicle-stimulating hormone, luteinizing hormone, and prolactin on follicular deoxyribonucleic acid synthesis in the hamster

Follicles were dissected by hand or enzymatically from the ovary of the proestrous hamster at 0900 h and classified into 10 stages: stages 1-4, follicles with 1-4 layers of granulosa cells and no theca; stages 5-8, preantral follicles with 5 or more layers of granulosa cells and theca to small antral follicles; stage 9, intermediate-sized atretic antral follicles; and stage 10, healthy preovulatory antral follicles. Follicles were then incubated for 2 h with [3H]thymidine [( 3H]Tdr) in the absence or presence of gonadotropins and with incorporation of radionuclide into DNA as the end point. FSH (25 ng) significantly stimulated [3H]Tdr incorporation in all stages of follicular development with a latency of 2 h, and this effect was inhibited by 2 micrograms unlabeled Tdr. While FSH and PRL (25 and 100 ng) stimulated [3H]Tdr incorporation in all stages, LH (0.2-5 ng) action began from stage 5 onward, when definitive thecal cells and LH receptors started appearing. LH (5 ng) also suppressed 25 ng FSH-induced DNA synthesis in stages 5-10; however, stages 1-4 were unaffected. Significant increases in both intra- and extracellular cAMP levels occurred in follicles at stages 2-10 after FSH administration. In contrast, LH was active in stages 5-10, whereas PRL was ineffective. Follicular DNA synthesis increased markedly when stimulated by 8-bromo-cAMP (0.01-2 mM). These results show that gonadotropins act directly as a primary stimulus at the level of small primary and secondary follicles to regulate DNA synthesis and, thus, perhaps the growth and differentiation of granulosa and thecal cells; cAMP functions as one of the possible intracellular mediators of gonadotropin action in initiating DNA replication.     

Opiate receptor subtypes in the rat hypothalamus and neurointermediate lobe

The potent opiate radioligands [3H]etorphine, [3H]ethylketocyclazocine (EKC), and [3H]naloxone, bound specifically and saturably to a single class of membrane-binding sites in rat neurointermediate lobe (NIL), with Kd values of 3.7, 24, and 51 nM, respectively. In the hypothalamus (Ht), [3H]etorphine bound to specific and saturable sites with a Kd of 2.9 nM. Binding-inhibition studies with [3H]etorphine and unlabeled etorphine-HCl as well as [3H]EKC and unlabeled EKC, revealed high and low affinity binding sites in rat Ht and NIL as well as in the neural lobe of the bovine pituitary gland. [3H]naloxone also bound specifically to two classes of sites in Ht membranes, but to only a single class of low affinity sites in NIL membranes. Specific binding represented 80-90% of total [3H]etorphine binding, about 75% of total [3H]EKC binding, and 45-55% of total [3H]naloxone binding at 22 C in NIL and Ht, respectively. Relative binding potencies derived from Ki values for binding-inhibition studies of [3H]etorphine with opioid peptides and opiates were: NIL, etorphine-HCl greater than dynorphin A greater than naloxone-HCl greater than dynorphin-(1-9) greater than beta-endorphin much greater than alpha-neoendorphin approximately (Leu5)enkephalin approximately DAGO (Tyr-D-Ala-Gly-NMe-Phe-Gly-ol); Ht, etorphine HCl greater than naloxone-HCl greater than beta-endorphin greater than dynorphin A much greater than DAGO greater than morphiceptin much greater than (Leu5)enkephalin. Specific [3H]etorphine binding was also demonstrable after preincubation of NIL membranes with DAGO and (Leu5)enkephalin and after preincubation of Ht membranes with morphiceptin and (Leu5)enkephalin; such binding could be displaced by nonradioactive dynorphin A. In addition, [3H]etorphine binding to bovine neural lobe was displaceable by naloxone-HCl, with an ED50 of 43 nM. Specific ligands for sigma-opiate receptors, such as (+)SKF 10,047 (N-allylnorcyclazocine), phencyclidine (PCP), and (-)cyclazocine, displaced specifically bound [3H]etorphine and [3H]EKC from NIL membranes only at high (micromolar) concentrations. However, specific [3H]PCP sites were of higher affinity in NIL and Ht membranes, with similar Kd values of 102 and 190 nM respectively, and different concentrations (0.15 and 1.32 pmol/mg protein, respectively). These data have revealed several differences in the opiate-binding properties of rat Ht and NIL membranes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Prepro-orexin and orexin receptor mRNAs are differentially expressed in peripheral tissues of male and female rats.

Orexins are produced specifically by neurons located in the lateral hypothalamus. Recent results suggested peripheral actions of orexins. Therefore, we analyzed the mRNA expression of prepro-orexin and the orexin receptor subtypes OX(1) and OX(2) in peripheral rat tissues. Using real-time quantitative RT-PCR we detected significant amounts of prepro-orexin mRNA in testis, but not in ovaries. OX(1) receptor mRNA was highly expressed in the brain and at lower levels in the pituitary gland. Only small amounts of OX(1) receptor mRNA were found in other tissues such as kidney, adrenal, thyroid, testis, ovaries, and jejunum. Very high levels of OX(2) receptor mRNA, 4-fold higher than in brain, were found in adrenal glands of male rats. Low amounts of OX(2) receptor mRNA were present in lung and pituitary. In adrenal glands, OX(2) receptor mRNA was localized in the zona glomerulosa and reticularis by in situ hybridization, indicating a role in adrenal steroid synthesis and/or release. OX(1) receptor mRNA in the pituitary and OX(2) receptor mRNA in the adrenal gland were much higher in male than in female rats. In the hypothalamus, OX(1) receptor mRNA was slightly elevated in female rats. The differential mRNA expression of orexin receptor subtypes in peripheral organs indicates discrete peripheral effects of orexins and the existence of a peripheral orexin system. This is supported by the detection of orexin A in rat plasma. Moreover, the sexually dimorphic expression of OX(1) and OX(2) receptors in the hypothalamus, pituitary, and adrenal glands suggests gender-specific roles of orexins in the control of endocrine functions.     

Localization and action of Dragon (repulsive guidance molecule b), a novel bone morphogenetic protein coreceptor, throughout the reproductive axis

Bone morphogenetic proteins (BMPs) play important roles in reproduction including primordial germ cell formation, follicular development, spermatogenesis, and FSH secretion. Dragon, a recently identified glycosylphosphatidylinositol-anchored member of the repulsive guidance molecule family, is also a BMP coreceptor. In the present study, we determined the tissue and cellular localization of Dragon in reproductive organs using immunohistochemistry and in situ hybridization. Among reproductive organs, Dragon was expressed in testis, epididymis, ovary, uterus, and pituitary. In the testis of early postnatal mice, Dragon was found in gonocytes and spermatogonia, whereas in immature testes, Dragon was only weakly expressed in spermatogonia. Interestingly, pregnant mare serum gonadotropin treatment of immature mice robustly induced Dragon production in spermatocytes. In adult testis, Dragon was found in spermatocytes and round spermatids. In the ovary, Dragon was detected exclusively within oocytes and primarily those within secondary follicles. In the pituitary, Dragon-expressing cells overlapped FSH-expressing cells. Dragon was also expressed in a number of cell lines originating from reproductive tissues including Ishikawa, Hela, LbetaT2, MCF-7, and JEG3 cells. Immunocytochemistry and gradient sucrose ultracentrifugation studies showed Dragon was localized in lipid rafts within the plasma membrane. In reproductive cell lines, Dragon expression enhanced signaling of exogenous BMP2 or BMP4. The present studies demonstrate that Dragon expression is dynamically regulated throughout the reproductive tract and that Dragon protein modulates BMP signaling in cells from reproductive tissues. The overlap between Dragon expression and the functional BMP signaling system suggests that Dragon may play a role in mammalian reproduction.