Characterization and localization of proopiomelanocortin messenger RNA in the adult rat testis.

Northern blot analysis of total RNA and polyadenylated RNA isolated from adult rat testes showed that a proopiomelanocortin (POMC)-like messenger RNA molecule is present in these extracts. The testicular POMC messenger RNA is comparable in length to amygdala and midbrain POMC messenger RNA and appears to be at least 200 nucleotides shorter than POMC messenger RNA found in the hypothalamus and anterior and intermediate lobes of the pituitary gland. Hybridization in situ showed that POMC messenger RNA is located in Leydig cells, which are the only testicular cells that contain immunostainable POMC-derived peptides. These results suggest that local synthesis of POMC occurs in the testis.

Retention of supraspinal delta-like analgesia and loss of morphine tolerance in delta opioid receptor knockout mice.

Gene targeting was used to delete exon 2 of mouse DOR-1, which encodes the delta opioid receptor. Essentially all 3H-[D-Pen2,D-Pen5]enkephalin (3H-DPDPE) and 3H-[D-Ala2,D-Glu4]deltorphin (3H-deltorphin-2) binding is absent from mutant mice, demonstrating that DOR-1 encodes both delta1 and delta2 receptor subtypes. Homozygous mutant mice display markedly reduced spinal delta analgesia, but peptide delta agonists retain supraspinal analgesic potency that is only partially antagonized by naltrindole. Retained DPDPE analgesia is also demonstrated upon formalin testing, while the nonpeptide delta agonist BW373U69 exhibits enhanced activity in DOR-1 mutant mice. Together, these findings suggest the existence of a second delta-like analgesic system. Finally, DOR-1 mutant mice do not develop analgesic tolerance to morphine, genetically demonstrating a central role for DOR-1 in this process.

Targeted deletion of the Vgf gene indicates that the encoded secretory peptide precursor plays a novel role in the regulation of energy balance.

To determine the function of VGF, a secreted polypeptide that is synthesized by neurons, is abundant in the hypothalamus, and is regulated in the brain by electrical activity, injury, and the circadian clock, we generated knockout mice lacking Vgf. Homozygous mutants are small, hypermetabolic, hyperactive, and infertile, with markedly reduced leptin levels and fat stores and altered hypothalamic proopiomelanocortin (POMC), neuropeptide Y (NPY), and agouti-related peptide (AGRP) expression. Furthermore, VGF mRNA synthesis is induced in the hypothalamic arcuate nuclei of fasted normal mice. VGF therefore plays a critical role in the regulation of energy homeostasis, suggesting that the study of lean VGF mutant mice may provide insight into wasting disorders and, moreover, that pharmacological antagonism of VGF action(s) might constitute the basis for treatment of obesity.

Retention of heroin and morphine-6 beta-glucuronide analgesia in a new line of mice lacking exon 1 of MOR-1.

Morphine produces analgesia by activating mu opioid receptors encoded by the MOR-1 gene. Although morphine-6 beta-glucuronide (M6G), heroin and 6-acetylmorphine also are considered mu opioids, recent evidence suggests that they act through a distinct receptor mechanism. We examined this question in knockout mice containing disruptions of either the first or second coding exon of MOR-1. Mice homozygous for either MOR-1 mutation were insensitive to morphine. Heroin, 6-acetylmorphine and M6G still elicited analgesia in the exon-1 MOR-1 mutant, which also showed specific M6G binding, whereas M6G and 6-acetylmorphine were inactive in the exon-2 MOR-1 mutant. These results provide genetic evidence for a unique receptor site for M6G and heroin analgesia.

Essential role of mu opioid receptor in the regulation of delta opioid receptor-mediated antihyperalgesia.

Analgesic effects of delta opioid receptor (DOR) -selective agonists are enhanced during persistent inflammation and arthritis. Although the underlying mechanisms are still unknown, membrane density of DOR was shown to be increased 72 h after induction of inflammation, an effect abolished in mu opioid receptor (MOR) -knockout (KO) mice [Morinville A, Cahill CM, Kieffer B, Collier B, Beaudet A (2004b) Mu-opioid receptor knockout prevents changes in delta-opioid receptor trafficking induced by chronic inflammatory pain. Pain 109:266-273]. In this study, we demonstrated a crucial role of MOR in DOR-mediated antihyperalgesia. Intrathecal administration of the DOR selective agonist deltorphin II failed to induce antihyperalgesic effects in MOR-KO mice, whereas it dose-dependently reversed thermal hyperalgesia in wild-type mice. The antihyperalgesic effects of deltorphin II were blocked by naltrindole but not d-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH(2) (CTOP) suggesting that this agonist was mainly acting through DOR. SNC80-induced antihyperalgesic effects in MOR-KO mice were also attenuated as compared with littermate controls. In contrast, kappa opioid receptor knockout did not affect deltorphin II-induced antihyperalgesia. As evaluated using mice lacking endogenous opioid peptides, the regulation of DOR's effects was also independent of beta-endorphin, enkephalins, or dynorphin opioids known to be released during persistent inflammation. We therefore conclude that DOR-mediated antihyperalgesia is dependent on MOR expression but that activation of MOR by endogenous opioids is probably not required.

Nociception increases during opioid infusion in opioid receptor triple knock-out mice.

Opioids are extensively used analgesics yet can paradoxically increase pain sensitivity in humans and rodents. This hyperalgesia is extensively conceptualized to be a consequence of opioid receptor activity, perhaps providing an adaptive response to analgesia, and to utilize N-methyl-D-aspartate (NMDA) receptors. These assumptions were tested here in opioid receptor triple knock-out (KO) mice lacking all three genes encoding opioid receptors (mu, delta, and kappa) by comparing their thermal nociceptive responses to the opioids morphine and oxymorphone with those of B6129F(1) controls. Injecting acute opioid bolus doses in controls caused maximal analgesia that was completely abolished in KO mice, confirming the functional consequence of the KO mouse opioid receptor deficiency. Continuous opioid infusion by osmotic pump in control mice also initially caused several consecutive days of analgesia that was shortly thereafter followed by several consecutive days of hyperalgesia. In contrast, continuously infusing KO mice with opioids caused no detectable analgesic response, but only immediate and steady declines in nociceptive thresholds culminating in several days of unremitting hyperalgesia. Finally, injecting the non-competitive NMDA receptor antagonist MK-801 during opioid infusion markedly reversed hyperalgesia in control but not KO mice. These data demonstrate that sustained morphine and oxymorphone delivery causes hyperalgesia independently of prior or concurrent opioid or NMDA receptor activity or opioid analgesia, indicating the contribution of mechanisms outside of current conceptions, and are inconsistent with proposals of hyperalgesia as a causative factor of opioid analgesic tolerance.

The absence of endogenous beta-endorphin selectively blocks phosphorylation and desensitization of mu opioid receptors following partial sciatic nerve ligation.

Phosphorylation of specific sites in the second intracellular loop and in the C-terminal domain have previously been suggested to cause desensitization and internalization of the mu-opioid receptor (MOP-R). To assess sites of MOP-R phosphorylation in vivo, affinity-purified, phosphoselective antibodies were raised against either phosphothreonine-180 in the second intracellular loop (MOR-P1) or the C-terminal domain of MOP-R containing phosphothreonine-370 and phosphoserine-375 (MOR-P2). We found that MOR-P2-immunoreactivity (IR) was significantly increased within the striatum of wild-type C57BL/6 mice after injection of the agonist fentanyl. Pretreatment with the antagonist naloxone blocked the fentanyl-induced increase. Furthermore, mutant mice lacking MOP-R showed only non-specific nuclear MOR-P2-IR before or after fentanyl treatment, confirming the specificity of the MOR-P2 antibodies. To assess whether MOP-R phosphorylation occurs following endogenous opioid release, we induced chronic neuropathic pain by partial sciatic nerve ligation (pSNL), which caused a significant increase in MOR-P2-IR in the striatum. pSNL also induced signs of mu opioid receptor tolerance demonstrated by a rightward shift in the morphine dose response in the tail withdrawal assay and by a reduction in morphine conditioned place preference (CPP). Mutant mice selectively lacking all forms of the beta-endorphin peptides derived from the proopiomelanocortin (Pomc) gene did not show increased MOR-P2-IR, decreased morphine antinociception, or reduced morphine CPP following pSNL. In contrast gene deletion of either proenkephalin or prodynorphin opioids did not block the effects of pSNL. These results suggest that neuropathic pain caused by pSNL in wild-type mice activates the release of the endogenous opioid beta-endorphin, which subsequently induces MOP-R phosphorylation and opiate tolerance.

Potentiation of opioid analgesia in dopamine2 receptor knock-out mice: evidence for a tonically active anti-opioid system.

Dopamine systems are intimately involved with opioid actions. Pharmacological studies suggest an important modulatory effect of dopamine and its receptors on opioid analgesia. We have now examined these interactions in a knock-out model in which the dopamine(2) (D(2)) receptor has been disrupted. Loss of D(2) receptors enhances, in a dose-dependent manner, the analgesic actions of the mu analgesic morphine, the kappa(1) agonist U50,488H and the kappa(3) analgesic naloxone benzoylhydrazone. The responses to the delta opioid analgesic [d-Pen(2),d-Pen(5)]enkephalin were unaffected in the knock-out animals. Loss of D(2) receptors also potentiated spinal orphanin FQ/nociceptin analgesia. Antisense studies using a probe targeting the D(2) receptor revealed results similar to those observed in the knock-out model. The modulatory actions of D(2) receptors were independent of final sigma receptor systems because the final sigma agonist (+)-pentazocine lowered opioid analgesia in all mice, including the D(2) knock-out group. Thus, dopamine D(2) receptors represent an additional, significant modulatory system that inhibits analgesic responses to mu and kappa opioids.

Altered processing of pro-orphanin FQ/nociceptin and pro-opiomelanocortin-derived peptides in the brains of mice expressing defective prohormone convertase 2.

The bioactivity of neuropeptides can be regulated by a variety of post-translational modifications, including proteolytic processing. Here, gene-targeted mice producing defective prohormone convertase 2 (PC2) were used to examine the post-translational processing of two neuroendocrine prohormones, pro-opiomelanocortin (POMC) and pro-orphanin FQ (pOFQ)/nociceptin (N), in the brain. Reversed-phase HPLC and gel-exclusion chromatography were combined with specific radioimmunoassays to analyze the processing patterns of these two prohormones in the hypothalamus and the amygdala. In the case of POMC, the lack of PC2 activity completely prevented carboxy-shortening of beta-endorphins and greatly diminished conversion of beta-lipotropin to gamma-lipotropin and beta-endorphin. Although conversion of beta-lipotropin to beta-endorphin decreased, the lack of PC2 activity caused an increase in beta-lipotropin and beta-endorphin levels in the mutant animals, but no increases in POMC or biosynthetic intermediates were seen. The extent of OFQ/N production was significantly lower in PC2-deficient mice and there was an accumulation of relatively large amounts of pOFQ/N and biosynthetic intermediates. These results demonstrate that PC2 is directly involved in the biogenesis of two brain neuropeptides in vivo and suggest that the specific prohormone and cellular context influences neuropeptide processing by PCs.

Increased gabaergic input to ventral tegmental area dopaminergic neurons associated with decreased cocaine reinforcement in mu-opioid receptor knockout mice.

There is general agreement that dopaminergic neurons projecting from the ventral tegmental area (VTA) to the nucleus accumbens and prefrontal cortex play a key role in drug reinforcement. The activity of these neurons is strongly modulated by the inhibitory and excitatory input they receive. Activation of mu-opioid receptors, located on GABAergic neurons in the VTA, causes hyperpolarization of these GABAergic neurons, thereby causing a disinhibition of VTA dopaminergic neurons. This effect of mu-opioid receptors upon GABA neurotransmission is a likely mechanism for mu-opioid receptor modulation of drug reinforcement. We studied mu-opioid receptor signaling in relation to cocaine reinforcement in wild-type and mu-opioid receptor knockout mice using a cocaine self-administration paradigm and in vitro electrophysiology. Cocaine self-administration was reduced in mu-opioid receptor knockout mice, suggesting a critical role of mu-opioid receptors in cocaine reinforcement. The frequency of spontaneous inhibitory post-synaptic currents onto dopaminergic neurons in the ventral tegmental area was increased in mu-opioid receptor knockout mice compared with wild-type controls, while the frequency of spontaneous excitatory post-synaptic currents was unaltered. The reduced cocaine self-administration and increased GABAergic input to VTA dopaminergic neurons in mu-opioid receptor knockout mice supports the notion that suppression of GABAergic input onto dopaminergic neurons in the VTA contributes to mu-opioid receptor modulation of cocaine reinforcement.

Localization of opioid receptor antagonist [3H]-LY255582 binding sites in mouse brain: comparison with the distribution of mu, delta and kappa binding sites.

Agonist stimulation of opioid receptors increases feeding in rodents, while opioid antagonists inhibit food intake. The pan-opioid antagonist, LY255582, produces a sustained reduction in food intake and body weight in rodent models of obesity. However, the specific receptor subtype(s) responsible for this activity is unknown. To better characterize the pharmacology of LY255582, we examined the binding of a radiolabeled version of the molecule, [(3)H]-LY255582, in mouse brain using autoradiography. In mouse brain homogenates, the K(d) and B(max) for [(3)H]-LY255582 were 0.156 +/- 0.07 nM and 249 +/- 14 fmol/mg protein, respectively. [(3)H]-LY255582 bound to slide mounted sections of mouse brain with high affinity and low non-specific binding. High levels of binding were seen in areas consistent with the known localization of opioid receptors. These areas included the caudate putamen, nucleus accumbens, claustrum, medial habenula, dorsal endopiriform nucleus, basolateral nucleus of the amygdala, hypothalamus, thalamus and ventral tegmental area. We compared the binding distribution of [(3)H]-LY255582 to the opioid receptor antagonist radioligands [(3)H]-naloxone (mu), [(3)H]-naltrindole (delta) and [(3)H]-norBNI (kappa). The overall distribution of [(3)H]-LY255582 binding sites was similar to that of the other ligands. No specific [(3)H]-LY255582 binding was noted in sections of mu-, delta- and kappa-receptor combinatorial knockout mice. Therefore, it is likely that LY255582 produces its effects on feeding and body weight gain through a combination of mu-, delta- and kappa-receptor activity.

Expression of the P450 side-chain cleavage and adrenodoxin genes begins during early stages of adrenal cortex development.

The steroid hormone products of the fetal adrenal cortex play an essential role in normal maturation of several organ systems during fetal development. In addition, adrenal steroids appear to play a local role in the establishment and maintenance of the chromaffin cells of the adrenal cortex. Despite these developmental roles of cortical steroids, little is known about when the cells of the fetal rat adrenal cortex begin to undergo biochemical differentiation into cells capable of producing steroid hormones and whether the timing of developmental changes in cortical properties is related to chromaffin cell differentiation. To investigate these problems, in situ hybridization and immunocytochemistry were used to examine the ontogeny of expression of both the P450 side-chain cleavage (P450scc) and adrenodoxin genes during rat development. Transcripts from both genes (but not P450c17) and the respective proteins encoded by them were detected specifically in the cells of the presumptive cortex as early as embryonic day 12 (e12), which is several days before the layered architecture of the adrenal cortex is established and the earliest age at which biochemical differentiation of these cells has been detected. The spatial and temporal expression patterns for both genes were similar over the period examined (e12-e16.5), and no heterogeneity of expression was observed among cortical cells. In addition, significant increases in the accumulation of P450scc and adrenodoxin mRNA transcripts occurred during the midgestational period, when the synthesis and secretion of ACTH from the fetal pituitary are increasing.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental regulation of proopiomelanocortin gene expression in the fetal and neonatal rat pituitary.

Expression of the POMC gene and secretion of its peptide products are under complex regulation in the pituitary by multiple factors. CRF stimulates POMC transcription and secretion in both adult anterior (AL) and intermediate (IL) pituitary lobes, whereas glucocorticoids have an inhibitory effect on POMC in the AL, but little, if any, effect in the IL. To determine when transcriptional responses elicited by these factors begin during development and whether they undergo changes during ontogeny, we used a solution hybridization/nuclease protection assay with a POMC exon 1-intron A splice junction probe to analyze simultaneously the levels of intron A-containing POMC heterogeneous nuclear RNA (hnRNA) and POMC mRNA in explant fetal and neonatal rat pituitaries. We examined responses to 8-bromo-cAMP, CRF, and dexamethasone (dex) at stages before and after innervation of the IL by dopaminergic neurons from the hypothalamus. Treatment of embryonic day 15 (e15) whole pituitaries with CRF (10(-7) M) for 1 h led to a 2.5-fold increase in the level of POMC hnRNA, while pretreatment with dex (10(-6) M) inhibited the CRF-induced stimulation of POMC transcription. These results demonstrate that by e15, POMC transcription is already responsive to both CRF and dex, and thus, functional receptors (coupled effectively to the POMC promoter) are present by this age. Initial studies of POMC mRNA levels at early postnatal ages showed that 1 mM 8-bromo-cAMP stimulated postnatal day 1 (p1) and p10 AL and neurointermediate lobe (NIL) POMC mRNA levels, and 10(-6) M dex inhibited this stimulation in p1 AL, p10 AL, and p1 NIL, but not in p10 NIL. These studies were extended to examine POMC hnRNA responses at these ages. Treatment with CRF for 1 h increased POMC hnRNA 1.9- and 1.5-fold in p1 and p10 AL, respectively, and pretreatment with dex blocked these CRF-mediated effects on AL POMC transcription. In the NIL on p1, CRF induced a 2-fold increase in POMC hnRNA, which (like that in the AL) was inhibited by 30-min pretreatment with dex; in contrast, on p10, dex did not affect the CRF-induced increase in POMC hnRNA. The glucocorticoid receptor subtype responsible for this effect was identified using treatments with specific agonist and antagonists. The type II receptor agonist RU 28362 had an effect similar to that of dex; at both 10(-6) and 10(-8) M, RU 28362 inhibited CRF-induced increases in POMC hnRNA in p1 NIL and p1 and p10 AL.(ABSTRACT TRUNCATED AT 400 WORDS)

Analysis of proopiomelanocortin gene expression during prenatal development of the rat pituitary gland.

The POMC gene is transcribed in 5-10% of adult rat anterior pituitary cells and nearly all intermediate lobe cells, but the end product peptide hormones produced by these two cell populations are distinct. We have here used in situ hybridization with 3H-POMC cRNA probes to investigate the temporal and spatial distribution of POMC mRNA containing cells during development of both lobes of the adenohypophysis. These hybridization patterns were, in addition, compared to the immunocytochemical distribution of POMC-related peptides on the same or adjacent sections. In both POMC populations, POMC-mRNA detection preceded POMC-derived peptide accumulation by at least 0.5 days. POMC-mRNA was first detected in the ventral aspect of Rathke's pouch (which will give rise to the anterior lobe) at e 13, which was two days earlier than POMC transcripts were first detected in the intermediate lobe; this result provides strong evidence that factors eliciting POMC transcription in these two populations act at distinct times during development. Further, since POMC mRNA containing cells were restricted to the anterior lobe during stages when intermediate lobe-like POMC peptides have been characterized biochemically, these results confirm immunocytochemical results and demonstrate that developmentally regulated alterations in the extent of POMC posttranslational processing occur during prenatal differentiation of anterior lobe POMC cells. POMC expression in the intermediate lobe was first observed in the midcaudal region in the midsagittal plane and subsequently extended in a gradient-like pattern to the rest of the intermediate lobe. Finally, semiquantitation of in situ hybridization signals documented that increases in the relative levels of POMC mRNA/cell occur at specific ages in both anterior and intermediate lobe. POMC cell populations and demonstrated that adult POMC mRNA/cell levels are not achieved before birth in either the anterior or intermediate lobe POMC cell populations.

The expression pattern of an insulin-like growth factor (IGF)-binding protein gene is distinct from IGF-II in the midgestational rat embryo.

Insulin-like growth factor-II (IGF-II), the predominant form of IGF in fetal and neonatal serum and tissues, is found in vivo complexed with IGF-binding proteins. One of these binding proteins, IGFBP-2, is present at high levels in fetal rat plasma and binds both IGF-I and IGF-II with high affinity. We here have used in situ hybridization to compare the distribution of IGFBP-2 mRNA with that of IGF-II mRNA in embryonic day 13.5-15 rat embryos. The spatial patterns of IGF-II and IGFBP-2 expression in the fetal trunk were distinct and, in general, nonoverlapping. Most mesoderm derivatives that express IGF-II at high levels contained little, if any, IGFBP-2 mRNA. Instead, IGFBP-2 mRNA was expressed at high levels in many cell types derived from ectoderm and endoderm. The expression of IGFBP-2 mRNA in the central nervous system (CNS) during this developmental period was examined in particular detail. The three most prominent sites of IGFBP-2 expression in the CNS were comprised of cells with nonneuronal phenotypes: 1) the epithelium of the choroid plexus, a tissue that produces cerebrospinal fluid; 2) the floor plate, an area that can guide axonal outgrowth from commissural neurons of the spinal cord in vitro; and 3) the infundibulum, the progenitor of the posterior pituitary that is believed to influence differentiation of the adjacent intermediate pituitary.(ABSTRACT TRUNCATED AT 250 WORDS)

Selective alterations in organ sizes in mice with a targeted disruption of the insulin-like growth factor binding protein-2 gene.

Insulin-like growth factor binding protein 2 (IGFBP-2) is one member of the family of IGF binding proteins believed to have both endocrine functions elicited by modulating serum IGF half-life and transport as well as autocrine/paracrine functions that result from blocking or enhancing the availability of IGFs to bind cell surface receptors. To clarify the in vivo role of IGFBP-2, we have used gene targeting to introduce a null IGFBP-2 allele into the mouse genome. Animals homozygous for the altered allele are viable and fertile, contain no IGFBP-2 mRNA, and have no detectable IGFBP-2 in the adult circulation. Heterozygous and homozygous animals showed no significant differences in prenatal or postnatal body growth. Analyses of organ weights in adult males, however, revealed that spleen weight was reduced and liver weight was increased in the absence of IGFBP-2. In addition, ligand blot analyses of sera from adult IGFBP-2 null males showed that IGFBP-1, IGFBP-3, and IGFBP-4 levels were increased relative to wild-type mice. These results demonstrate that up-regulation of multiple IGFBPs accompanies the absence of IGFBP-2 and that IGFBP-2 has a critical role, either directly or indirectly, in modulating spleen and liver size.

Developmental changes in levels of proopiomelanocortin intron A-containing heterogeneous nuclear RNA and mature messenger RNA in the anterior and neurointermediate lobes of the rat pituitary.

The POMC cells of the rat pituitary undergo dynamic phenotypic changes during differentiation. Here we have determined that alterations in the relative levels of a POMC precursor RNA species and POMC mRNA occur during development and may represent another level at which the POMC phenotype is developmentally regulated. We performed solution hybridization/nuclease protection assays using a POMC exon 1/intron A splice junction probe to quantitate levels of both intron A-containing POMC heterogeneous nuclear (hnRNA) and fully processed POMC mRNA in separated anterior and neurointermediate lobes of the fetal, neonatal, and adult pituitary. The levels of POMC hnRNA per anterior lobe increased 7-fold from embryonic day 15 to adulthood (0.022 to 0.159 fmol/lobe), while POMC mRNA levels increased 121-fold (0.15 to 18.2 fmol/lobe). POMC hnRNA levels per neurointermediate lobe increased 23-fold from embryonic day 18 to adulthood (0.024 to 0.54 fmol/lobe), while POMC mRNA levels increased 69-fold (0.65 to 44.6 fmol/lobe). Thus, both anterior and neurointermediate lobes contain higher relative abundances of POMC hnRNA compared to mRNA during early development. These subsequently decrease (from 1:7 to 1: approximately 110 in the anterior lobe and from 1:27 to 1:83 in the intermediate lobe over the ages examined) as the levels of POMC mRNA in both anterior and neurointermediate lobe increase at a greater rate than POMC hnRNA as development progresses. These results provide the first measurements of POMC mRNA and hnRNA levels during ontogeny and suggest that there may be a developmental change in the regulation of POMC primary transcript processing.

The embryonic pattern of rat insulin-like growth factor-I gene expression suggests a role in induction and early growth of the liver.

The classic experiments of LeDouarin and her colleagues demonstrated that the mesenchymal cells of the septum transversum induce both the rapid proliferation of the adjacent endodermally-derived foregut cells and their differentiation into the hepatocyte component of the liver. We have used in situ hybridization to test whether this inductive activity correlates with the developmental expression of genes encoding insulin-like growth factors-I and -II (IGF-I and -II) and/or proteins that may mediate IGF action (IGF binding protein-2 and the IGF-I receptor). We found that the onset of expression of the transcript for IGF-I in the septum transversum correlates precisely with the time that the inductive activity appears. In fact, the septum transversum is the earliest site of IGF-I mRNA expression detectable in the rat embryo by in situ hybridization.

Stimulation of beta-endorphin secretion by corticotropin-releasing factor in primary rat Leydig cell cultures.

CRF, a hypothalamic peptide, is a potent stimulator of POMC synthesis and secretion in the pituitary. POMC biosynthesis has been documented in the testis, specifically in Leydig cells, and recent studies suggest that CRF is synthesized locally in the testis. A reverse hemolytic plaque assay and immunocytochemistry with Leydig cell-specific antibodies were used to study the effect of CRF on secretion of the POMC peptide beta-endorphin (beta EP) from normal rat primary Leydig cell cultures. In enriched Leydig cell preparations incubated with beta EP antiserum (diluted 1:50) then with complement (diluted 1:25), approximately 15% of immunocytochemically identified Leydig cells formed plaques. Preabsorption of the antiserum with beta EP (2 micrograms/microliters antiserum) overnight at 4 C abolished the formation of plaques. Increasing concentrations of CRF (from 10(-1) to 10(-7) M) resulted in an approximately 80% increase in both the percentage of plaque-forming cells and the mean plaque size. When the CRF antagonist CRF-(9-41) (10(-6) M) was added in the presence of CRF, the increases in plaque number and average size did not occur. These results demonstrate that Leydig cells have functional CRF receptors and that beta EP secretion from these cells is stimulated by CRF.

Distinct expression patterns of insulin-like growth factor binding proteins 2 and 5 during fetal and postnatal development.

Insulin-like growth factors (IGFs), when isolated from serum or tissue fluids, are usually found as part of a protein complex which also contains one of several IGF binding proteins (IGFBPs). Although some IGFBPs have been shown to alter interactions of IGFs with their receptors in vitro and can modify the responses of cultured cells to exogenous IGFs, the in vivo functions of IGFBPs remain unclear. This study examines expression of a recently described IGFBP gene, IGFBP-5, in the rat embryo and fetus and in selected adult tissues. Embryonic IGFBP-5 messenger RNA (mRNA) can be detected as early as embryonic day 10.5 and has an mRNA expression pattern distinct from the previously characterized pattern of IGFBP-2 mRNA expression. Major sites of IGFBP-5 expression during early postimplantation stages of development include the notochord, the floor plate, regions of the surface ectoderm, muscle precursor cells, and specific axial regions of neuroepithelium. Later in development IGFBP-5 mRNA is found in several regions of the central nervous system, including the proliferative zone of the external granule layer of the cerebellum and the mitral neurons of the olfactory bulb, as well as in muscle precursor populations of the developing limb, and in most cells of the anterior pituitary. In addition, only a subset of pituicytes in the adult posterior pituitary express IGFBP-5, which provides the first evidence that this cell population is biochemically heterogeneous. Taken together, these data suggest functions for IGFBP-5 during development of several organ systems.