Colocalization Of mRNA Research Articles

Asymmetrical localization of mRNAs in enterocytes of human jejunum.

Intracellular localization of specific mRNAs is known to be a mechanism for targeting proteins to specific sites within the cell. Previous studies from this laboratory have demonstrated co-localization of mRNAs and proteins for a number of genes in absorptive enterocytes of fetal rat intestine. The present study was undertaken to examine in human enterocytes the intracellular localization patterns of mRNAs for the microvillous membrane proteins lactase-phlorizin hydrolase (LPH), sucrase-isomaltase (SI), and intestinal alkaline phosphatase (IAP), and the cytoskeletal protein beta-actin. In sections of human jejunum, mRNAs were localized by in situ hybridization using digoxigenin-labeled anti-sense RNA probes. Both LPH and SI mRNAs were localized to the apical region of villous enterocytes, whereas IAP and beta-actin mRNAs were detected both apically and basally relative to the nucleus. Therefore, in contrast to LPH, SI, and beta-actin mRNAs, which co-localize with their encoded proteins, that of IAP is present in the basal region of the cell where IAP protein has not directly been demonstrated to be present. Absorptive enterocytes from humans possess the mechanisms for intracellular mRNA localization, but not all mRNAs co-localize with their encoded proteins.

MT1-MMP and MMP-2 mRNA expression in human ovarian tumors: Possible implications for the role of desmoplastic fibroblasts

Expression of activated MMP-2 (72 kDa type IV collagenase) is highly associated with the malignant phenotype in adenocarcinomas, but predominant expression of the mRNA appears to be in stromal cells. MT1-MMP (membrane type 1-matrix metalloproteinase) is implicated in tumor-epithelial cell surface activation of latent proMMP-2, indicating a mechanism for tumor-stromal interaction in invasion. We determined the relative mRNA distribution of these MMPs in human ovarian tumors with a view to analyzing potential variations in the epithelial-mesenchymal interactions dictating ovarian tumor cell spread. In situ hybridization using 35S-labeled riboprobes was used to analyze 33 human ovarian tumors and mouse xenografts of human ovarian (DOV 13, SKOV3) and breast (MCF7) tumor cell lines known to express MT1-MMP and MMP-2. MMP-2 mRNA was expressed in 31 of 33 and MT1-MMP mRNA was expressed in 29 of 33 tumor cases. MMP-2 mRNA was predominantly expressed in desmoplastic fibroblasts and in the subepithelial stroma. MT1-MMP mRNA showed some colocalization with MMP-2 in stromal cells. Neoplastic epithelial cell labeling for MT1-MMP mRNA was present in border-line and malignant tumors but not in benign tumors, and was invariably less than stromal labeling. Xenografts of DOV 13, SKOV 3, and MCF 7 cells showed some stromal localization of MMP-2 mRNA and weak labeling of DOV 13 cells. There was variable labeling for MT1-MMP mRNA in the neoplastic cells only. The colocalization of MT1-MMP and MMP-2 mRNAs in ovarian carcinoma stroma supports the view that MT1-MMP is closely associated with MMP-2 expression and function. It suggests that either additional mechanisms are involved in regulating MMP-2 activation at the tumor cell surface, or more intriguingly, that desmoplastic fibroblasts may be the primary mediators of extracellular matrix remodeling with respect to this system.

Regional and cellular distribution of serotonin 5-hydroxytryptamine2a receptor mRNA in the nucleus accumbens, olfactory tubercle, and caudate putamen of the rat

This paper describes the regional and cellular distribution of serotonin 5-hydroxytryptamine2a (5-HT2a) receptor mRNA in (sub)regions of the rat striatum by using in situ hybridization. Our results indicate that 5-HT2a mRNA is distributed heterogeneously in this brain region. Regional densitometry of autoradiograms from striatal sections hybridized with isotope-labeled cRNA probes showed that mRNA levels were highest in the olfactory tubercle, lower in the nucleus accumbens, and lowest in the caudate-putamen. In the nucleus accumbens, the average mRNA levels in the shell were higher than those in the core. These data suggest a particular relevance for the 5-HT2a receptor for olfactory tubercle- and shell-related functions. Therefore, in the nucleus accumbens and the olfactory tubercle, the cellular localization of 5-HT2a mRNA was investigated by determining the colocalization of 5-HT2a mRNA with enkephalin mRNA or dynorphin mRNA. 5-HT2a mRNA was found in enkephalinergic as well as dynorphinergic neurons. Thus, there does not seem to be a differential distribution of this receptor in the output routes of the ventral striatum. In all of the subregions investigated (core, medial shell, and lateral shell of the nucleus accumbens and the olfactory tubercle), only subpopulations of the total enkephalinergic and dynorphinergic populations were found to contain 5-HT2a mRNA. For enkephalin, the percentage colocalization was higher in the lateral shell (61%) compared with the other subregions (38-45%). For dynorphin, the percentage colocalization was higher in the olfactory tubercle (68%) than in the other subregions (34-43%). The differences in (sub)regional mRNA levels and in colocalization with opioids suggest a considerable regional differentiation in the effects of 5-HT2a-mediated neurotransmission in the striatum.

Regional and cellular distribution of serotonin 5‐hydroxytryptamine2a receptor mRNA in the nucleus accumbens, olfactory tubercle, and caudate putamen of the rat

This paper describes the regional and cellular distribution of serotonin 5-hydroxytryptamine2a (5-HT2a) receptor mRNA in (sub)regions of the rat striatum by using in situ hybridization. Our results indicate that 5-HT2a mRNA is distributed heterogeneously in this brain region. Regional densitometry of autoradiograms from striatal sections hybridized with isotope-labeled cRNA probes showed that mRNA levels were highest in the olfactory tubercle, lower in the nucleus accumbens, and lowest in the caudate-putamen. In the nucleus accumbens, the average mRNA levels in the shell were higher than those in the core. These data suggest a particular relevance for the 5-HT2a receptor for olfactory tubercle- and shell-related functions. Therefore, in the nucleus accumbens and the olfactory tubercle, the cellular localization of 5-HT2a mRNA was investigated by determining the colocalization of 5-HT2a mRNA with enkephalin mRNA or dynorphin mRNA. 5-HT2a mRNA was found in enkephalinergic as well as dynorphinergic neurons. Thus, there does not seem to be a differential distribution of this receptor in the output routes of the ventral striatum. In all of the subregions investigated (core, medial shell, and lateral shell of the nucleus accumbens and the olfactory tubercle), only subpopulations of the total enkephalinergic and dynorphinergic populations were found to contain 5-HT2a mRNA. For enkephalin, the percentage colocalization was higher in the lateral shell (61%) compared with the other subregions (38–45%). For dynorphin, the percentage colocalization was higher in the olfactory tubercle (68%) than in the other subregions (34–43%). The differences in (sub)regional mRNA levels and in colocalization with opioids suggest a considerable regional differentiation in the effects of 5-HT2a-mediated neurotransmission in the striatum. J. Comp. Neurol. 389:1–11, 1997. © 1997 Wiley-Liss, Inc.

Angiotensin II AT1AReceptor mRNA Expression Is Induced by Estrogen–Progesterone in Dopaminergic Neurons of the Female Rat Arcuate Nucleus

Brain angiotensin II (Ang II) inhibits pituitary prolactin release by an indirect mechanism requiring stimulation of dopamine formation and release. We report that [125I]Sar1-Ang II binding to AT1 receptors and AT1A receptor mRNA expression increase selectively in the dorsomedial arcuate nucleus of 17beta-estradiol-primed ovariectomized rats after treatment with progesterone. In hormone-treated rats, arcuate nucleus AT1A receptor mRNA expression is associated with tyrosine hydroxylase-positive neurons. No AT1A receptor mRNA was detected in tyrosine hydroxylase-positive cells of the arcuate nucleus of intact male rats. Conversely, in the anterior pituitary, where local or circulating Ang II stimulates prolactin release, [125I]Sar1-Ang II binding to AT1 receptors and AT1B receptor mRNA expression are decreased in 17beta-estradiol/progesterone-treated ovariectomized rats. Thus, AT1A receptors in the dorsal arcuate nucleus and AT1B receptors in the anterior pituitary are regulated inversely by estrogen/progesterone treatment, supporting the hypothesis of a dual role for brain and pituitary Ang II on prolactin release. The colocalization of AT1A receptor mRNA and tyrosine hydroxylase in neurons of the arcuate nucleus furthermore indicates that within this area central Ang II acts directly on dopaminergic neurons. These results support the hypothesis that central Ang II inhibits pituitary prolactin release indirectly via modulation of dopaminergic activity in the arcuate nucleus.

Codistribution of nicotinic acetylcholine receptor subunit alpha3 and beta4 mRNAs during rat brain development.

We have used in situ hybridization to characterize the ontogeny of alpha3 and beta4 nicotinic acetylcholine receptor (nAChR) subunit mRNA expression in rat brain. Transcripts for both subunits were detected in embryonic brain, although overlapping expression of alpha3 mRNA was only evident in areas of strong beta4 mRNA expression, including the medial habenula, locus coeruleus, the cerebellar primordium, and several motor and sensory brainstem nuclei. During the perinatal period, the independent expression of alpha3 mRNA declined, and greater correspondence in the temporal and spatial expression of alpha3 and beta4 subunit mRNAs emerged. In general, beta4 mRNA expression preceded that of alpha3 mRNA by 1 to 2 days. Overlapping expression patterns were transiently detected in the caudate putamen, basal forebrain, frontal and visual cortices, and in the CA3 field of hippocampus. Codistribution that lasted throughout development and into adulthood was noted in a number of brain areas, including the retrosplenial cortex, subiculum, medial habenula, interpeduncular nucleus, locus coeruleus, and brainstem motor nuclei. In many of these regions, alpha5 subunit mRNA was also expressed. Colocalization of alpha3 and beta4 mRNAs with choline acetyltransferase mRNA was detected in cholinergic neurons of the brainstem motor nuclei, nucleus ambiguus, dorsal motor nucleus of the vagus, motor trigeminal nucleus, and facial nucleus, but not in most forebrain cholinergic cells. The extensive correspondence in temporal and spatial distribution of alpha3 and beta4 mRNAs throughout postnatal brain development suggests that these subunits may be coordinately regulated and may form functional neuronal nAChRs with significant developmental roles.

Expression of Angiotensin Type-1 (AT1) and Type-2 (AT2) Receptor mRNAs in the Adult Rat Brain: A Functional Neuroanatomical Review

The discovery that all components of the renin–angiotensin system (RAS) are present in the central nervous system led investigators to postulate the existence of a local brain RAS. Supporting this, angiotensin immunoreactive neurons have been visualized in the brain. Two major pathways were described: a forebrain pathway which connects circumventricular organs to the median preoptic nucleus, paraventricular nucleus, and supraoptic nucleus, and a second pathway connecting the hypothalamus to the medulla oblongata. Blood–brain barrier deficient circumventricular organs are rich in angiotensin II receptors. By activating these receptors, circulating angiotensin II may act on central cardiovascular centers via angiotensinergic neurons, providing a link between peripheral and central angiotensin II systems. Among the effector peptides of the brain RAS, angiotensin II and angiotensin III have the same affinity for the two pharmacologically well-defined receptors: type 1 (AT1) and type 2 (AT2). When injected in the brain, these peptides increase blood pressure, water intake, and anterior and posterior pituitary hormone release and may modify memory and learning. The cloning of AT1 and AT2 receptor cDNAs has revealed that these receptors belong to the seven transmembrane domain receptor family. In rodents, two AT1 receptor subtypes, AT1A and AT1B, have been isolated. Using specific riboprobes forin situhybridization histochemistry, recent studies mapped the distribution of AT1A, AT1B, and AT2 receptor mRNAs in the adult rat and found a predominant expression of AT1A and AT2 mRNA in the brain and of AT1B in the pituitary. Very limited overlap was found between the brain expression of AT1A and AT2 mRNAs. In several functional entities of the brain, such as the preoptic region, the hypothalamus, the olivocerebellary system, and the brainstem baroreflex arc, the colocalization of receptor mRNA, binding sites, and angiotensin immunoreactive nerve terminals suggests local synthesis and expression of angiotensin II receptors. In other areas, such as the bed nucleus of the stria terminalis, the median eminence, or certain parts of the nucleus of the solitary tract, angiotensin II receptors are likely of extrinsic origin. The neuronal expression of AT1A and AT2 receptors was demonstrated in the subfornical organ, the hypothalamus, and the lateral septum. By using double labelin situhybridization, AT1A receptor expression was localized in corticotropin releasing hormone but not in vasopressin containing neurons in the hypothalamus. The information is discussed together with functional data concerning the role of brain angiotensins, in an attempt to provide a better understanding of the physiological and functional roles of each receptor subtype.

Co-localization of preprosomatostatin mRNA and preprotachykinin A mRNA in neurons of the rat suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN), a circadian oscillator, contains various peptides arranged in the compartment-specific manner. Somatostatin (SS) and substance P (SP), a peptide derived from preprotachykinin A (PPT-A), are expressed in neurons in the intermediate zone, a narrow area between the major dorsomedial and ventrolateral subdivisions. In the present study, we examined the possibility of co-localization of SS and SP in the SCN by a double-labeling in situ hybridization method using 35S-labeled and digoxigenin-labeled cRNA probes. In the SCN, most of preprosomatostatin (PPSS) mRNA-containing neurons expressed PPT-A mRNA (86%) and, in turn, almost all preprotachykinin A (PPT-A) mRNA-expressing neurons expressed PPSS mRNA signals (97%). Both PPSS and PPT-A mRNAs were also detected in the cerebral cortex and the caudate-putamen, however, their co-existence was extremely rare (< 4%) in these regions. Since the pharmacological effects of SS and SP are similar to that of the light pulses exposed on animals under constant darkness, the co-release of peptides might be an important process for entraining the circadian clock in the SCN.

Effect of immune and metabolic challenges on the luteinizing hormone-releasing hormone neuronal system in cycling female rats: an evaluation at the transcriptional level.

The purpose of this study was to investigate the influence of immune (systemic endotoxin administration) and metabolic (fasting) challenges on LHRH neuronal activity and transcription in the organum vasculosum of the lamina terminalis/medial preoptic area as well as on the expression of the LHRH receptor (LHRH-R) in the anterior pituitary of cycling female rats. The reproductive stages of adult female rats (200-250 g; 14 h of light; lights on at 0600 h) were verified by daily vaginal smears taken every morning for a minimum of three or four cycles before the experiment. The acute-phase response was induced via an i.p. injection of lipopolysaccharide (LPS; 200 microg/100 g BW), whereas the metabolic challenge consisted of food deprivation for at least 48 h. Control and challenged rats were killed at specific times in the ovulatory cycle (1200, 1500, and 1800 h on proestrus and diestrous day 2). Frozen brains and pituitaries were mounted on a microtome, cut into 30-microm slices, and then processed for the detection of transcripts encoding either LHRH or LHRH-R by means of in situ hybridization histochemistry using intronic (heteronuclear RNA) and exonic [messenger RNA (mRNA)] riboprobes. Dual immunocytochemistry to detect Fos-immunoreactive (ir) nuclei in LHRH-ir perikarya and colocalization of LHRH mRNA with Fos protein during the day of proestrus were performed by using both in situ hybridization and immunocytochemistry techniques on the same brain sections. The percentage of LHRH-ir and LHRH-expressing neurons displaying positive Fos-ir nuclei during the afternoon of proestrus was significantly inhibited 3 h after endotoxin administration. Rats exhibited an increase in the levels of LHRH primary transcript in the organum vasculosum of the lamina terminalis/medial preoptic area structure at 1500 h on proestrus, a phenomenon significantly attenuated by LPS injection only at this phase of the estrous cycle. On the other hand, fasting did not affect LHRH neuronal activity or gene expression in intact cycling rats, but affected these cells in animals exhibiting a disruption of the ovulatory cycle. Interestingly, LPS caused a profound down-regulation of LHRH-R gene expression in the anterior pituitary throughout the entire estrous cycle. Although food deprivation provoked a more variable pattern of LHRH-R mRNA in cycling rats, the signal for this transcript in the adenohypophysis was deeply altered in those showing a perturbed cycle. These results provide evidence that immune challenge interferes with the LHRH system at both hypothalamic and pituitary levels, whereas alteration of that neuroendocrine system in food-deprived rats seems highly associated with the impairment of reproductive cyclicity.

Co-localization of alpha1D adrenergic receptor mRNA with mineralocorticoid and glucocorticoid receptor mRNA in rat hippocampus.

Many adaptive changes occur in response to chronic or repeated stress, involving complex regulatory interactions between central stress-related afferents and the central components of the hypothalamo-pituitary-adrenal (HPA) axis. One change associated with chronic stress is an attenuation of corticosteroid receptor-mediated feedback inhibition of the HPA axis, a process thought to involve corticosteroid receptors in the hippocampus. A prominent stress-related central afferent that innervates the hippocampus and that may participate in the regulation of the HPA axis is the central noradrenergic system. Previous evidence suggests that alpha1 adrenergic receptors may down-regulate hippocampal corticosteroid receptors, and may thus contribute to stress-induced facilitation of HPA responses. In the present study, we used combined nonisotopic and radioisotopic in situ hybridization to examine the overlapping expression and co-localization of mRNA encoding the post-synaptic alpha1D adrenergic receptor subtype, the major alpha1 subtype expressed in hippocampus, with mRNA for the two corticosteroid receptor subtypes, type I (mineralocorticoid receptor, MR) and type II (glucocorticoid receptor, GR) in rat hippocampal neurons. We observed overlapping distributions and an extensive degree of co-localization of alpha1D receptor mRNA with both corticosteroid receptor subtype messages, establishing an anatomical substrate by which these two receptor systems may directly regulate each other. The potential interaction between co-localized adrenergic and corticosteroid receptors in hippocampus may contribute to stress-induced alterations in the HPA response to subsequent stress.

CNTF induces GFAP in a S-100α brain cell population: the pattern of CNTF-αR suggests an indirect mode of action

In a recent report, we demonstrated that intracerebral injections of the pleiotropic cytokine, ciliary neurotrophic factor (CNTF), into developing postnatal rats evoked a severe inflammatory response as determined by the appearance of reactive astrocytes and activated microglia. Considering the likely involvement of CNTF in the injury response, we felt it was important to further understand the role of CNTF in the developing rat CNS. In this study, we examined the responsiveness of other cell populations to intracerebral injections of CNTF. We report that CNTF increases glial fibrillary acidic protein (GFAP), while having no appreciable effect on the levels of other intermediate filaments including vimentin and neurofilament. Moreover, CNTF did not affect the expression of the mature oligodendrocyte gene, myelin basic protein. These results suggest that CNTF is highly specific in its regulation of GFAP. In our previous study, we showed CNTF to increase GFAP in a cell population that already exists in the CNS parenchyma. To determine the origin of the CNTF-induced reactive astrocytes, therefore, we have utilized a technique of combined in situ hybridization and immunocytochemistry. To examine the possibility that CNTF acts on oligodendrocyte precursors to give rise to reactive astrocytes, the platelet-derived growth factor alpha receptor (PDGF-αR) was utilized as a riboprobe in conjunction with an antibody to GFAP. Examination of CNTF-induced GFAP + astrocytes revealed no colocalization with PDGF-αR mRNA. In contrast, when we utilized an S100α antibody recognizing a calcium binding protein in immature astrocytes, we found colocalization of S100α and GFAP mRNA. These data suggest that CNTF induces an upregulation of GFAP in immature S100α + astrocytes. Examination of the CNTF-α receptor mRNA revealed no change in expression following CNTF treatment. Unexpectedly, however, the CNTF-induced astrogliotic response appears to be indirect since the CNTF-α receptor was solely expressed by neurons in the cytokine-treated animals.

Expression of mRNA for brain-derived neurotrophic factor in the dorsal root ganglion following peripheral inflammation

It is well known that the nerve growth factor (NGF) may serve as a link between inflammation and hyperalgesia. Recent experiments showed that systemic injection of NGF dramatically stimulated the expression of brain-derived neurotrophic factor (BDNF) mRNA in the dorsal root ganglion (DRG). In the present study, we evaluated the change of BDNF mRNA in the DRG following peripheral inflammation and also observed colocalization of BDNF and trkA mRNAs by means of in situ hybridization histochemistry in rats. Peripheral tissue inflammation produced by an intraplantar injection of Freund's adjuvant into the paws significantly increased BDNF mRNA levels in the DRG and many neurons expressing trkA mRNA showed increased expession of BDNF mRNA. Intraplantar injection of antibody to NGF together with Freund's adjuvant prevented the increase in BDNF mRNA. These findings suggest that peripheral inflammation induces an increased expression of BDNF mRNA which is mediated by NGF in DRG.

Localization studies of IGFBP-2 and IGFBP-5 in the anterior compartment of the eye

Purpose. Insulin-like growth factor binding proteins (IGFBPs) may modulate insulin-like growth factor-I (IGF-I) action and are important regulating factors in the IGF system. Our aim was to determine the presence of IGFBP-2 and -5 in the anterior compartment of the eye and to compare the histological sites of these IGFBP proteins with the respective IGFBP mRNAs. Methods. To investigate this, immunohistochemistry was used to detect the presence of IGFBP-2 and -5 proteins, and in situ hybridization was used to determine the presence of IGFBP-2 and -5 mRNAs. The studies were performed in normal adult male Sprague-Dawley rats. Immunohistochemistry was performed by the immunoperoxidase method with polyclonal anti-bovine-IGFBP-2 and antihuman-IGFBP-5 antibodies. In situ hybridization was performed using 35 S-radiolabelled riboprobes. Results. IGFBP-2 mRNA and protein were demonstrated in the outer non-pigmented ciliary epithelium, the corneal germinal epithelium and the corneal endothelium. IGFBP-2 mRNA was detected in these same histological layers of the ciliary processes and the cornea. IGFBP-5 mRNA localized to the stroma and also to the inner pigmented ciliary epithelium and IGFBP-5 protein was demonstrated in the adjacent outer non-pigmented ciliary epithelium. IGFBP-5 mRNA and protein were not demonstrated in the cornea. IGFBPs-2 and -5 were not demonstrated by immunohistochemistry in other structures of the eye. Conclusion. We have shown co-localization of IGFBP-2 mRNA and protein and adjacent cellular localization of IGFBP-5 mRNA and proteins in the anterior compartment of the eye. The presence of IGFBP-2 and -5 in the outer ciliary epithelium suggests secretion into the aqueous humour, where they may enhance trapping of IGF-I which may be important for lens and corneal cell survival. Our studies outlining the site of locally synthesized IGFBPs suggests specific roles in regulation of IGFs and highlights the potential importance of the IGF system in the eye.

GLAST mRNA expression in the periventricular area of experimental hydrocephalus.

Glutamate transporters play an important role in maintaining the extracellular glutamate concentration below the neurotoxic level. We investigated the expression of glutamate/aspartate transporter (GLAST) mRNA in the periventricular region of rats with kaolin-induced hydrocephalus by in situ hybridization (ISH). The density of GLAST mRNA-positive cells and the level of hybridization signals per positive cell significantly increased in the acute stage of hydrocephalus. We also demonstrated co-localization of GLAST mRNA and GFAP immunoreactivity in a single cell using the combined methods of ISH and immunohistochemistry. These findings suggest that GLAST is expressed in the reactive astrocytes of the periventricular area and regulates extracellular glutamate concentration after hydrocephalic brain injury.

Regulation of striatal D1A dopamine receptor gene transcription by Brn-4.

Brn-4 is a member of the POU transcription factor family and is expressed in the central nervous system. In this study, we addressed whether Brn-4 regulates expression of the D1A dopamine receptor gene. We found a functional Brn-4 responsive element in the intron of this gene by means of cotransfection chloramphenical acetyltransferase assays. This region contains two consensus sequences for binding of POU factors. Gel mobility-shift assays using glutathione S-transferase-Brn-4 fusion protein indicated that Brn-4 binds to these sequences. Both these sites are essential for transactivation by Brn-4 because deletion of either significantly reduced this enhancer activity. In situ hybridization revealed colocalization of Brn-4 and D1A mRNAs at the level of a single neuron in the rat striatum where this dopamine receptor is most abundantly expressed. Gel mobility-supershift assay using rat striatal nuclear extract and Brn-4 antibody confirmed the presence of Brn-4 in this brain region and its ability to bind to its consensus sequences in the D1A gene. These data suggest a functional role for Brn-4 in the expression of the D1A dopamine receptor gene both in vitro and in vivo.

Co-expression of steroid hormone receptors in opioid peptide-containing neurons correlates with patterns of gene expression during the estrous cycle.

The anteroventral periventricular nucleus (AVPV) of the preoptic region represents an essential component of neural pathways regulating gonadotropin secretion, and contains sexually dimorphic populations of neurons that express dynorphin or enkephalin. In the present study we used in situ hybridization to measure prodynorphin (PDYN) and proenkephalin (PENK) mRNA in the AVPV of intact animals killed on each day of the cycle. Levels of PDYN mRNA were lowest in animals killed on the afternoon of proestrus and then increased by over 60% by the morning of the following day. Expression of PENK mRNA was generally stable during the cycle, but a small yet significant reduction was detected on proestrus relative to levels of PENK mRNA in animals killed on the day of diestrus. In addition, we used double in situ hybridization to demonstrate that the majority of PDYN mRNA-containing neurons express both estrogen (50%) and progesterone receptor (85%) mRNAs. Only one quarter of the PENK-containing neurons also co-express estrogen receptor mRNA, and fewer than 10% of the PENK mRNA neurons express PR mRNA. Thus, the differential expression of PDYN and PENK during the cycle generally correlates with distinct differences in the degree of colocalization of ER and PR mRNA in PDYN and PENK mRNA-containing neurons in the AVPV.

Acidic fibroblast growth factor mRNA is expressed by basal forebrain and striatal cholinergic neurons.

Evidence for the importance of the basal forebrain cholinergic system in the maintenance of cognitive function has stimulated efforts to identify trophic mechanisms that protect this cell population from atrophy and dysfunction associated with aging and disease. Acidic fibroblast growth factor (aFGF) has been reported to support cholinergic neuronal survival and has been localized in basal forebrain with the use of immunohistochemical techniques. Although these data indicate that aFGF is present in regions containing cholinergic cell bodies, the actual site of synthesis of this factor has yet to be determined. In the present study, in situ hybridization techniques were used to evaluate the distribution and possible colocalization of mRNAs for aFGF and the cholinergic neuron marker choline acetyltransferase (ChAT) in basal forebrain and striatum. In single-labeling preparations, aFGF mRNA-containing neurons were found to be codistributed with ChAT mRNA+ cells throughout all fields of basal forebrain, including the medial septum/diagonal band complex and striatum. By using a double-labeling (colormetric and isotopic) technique, high levels of colocalization (over 85%) of aFGF and ChAT mRNAs were observed in the medial septum, the diagonal bands of Broca, the magnocellular preoptic area, and the nucleus basalis of Meynert. The degree of colocalization was lower in the striatum, with 64% of the cholinergic cells in the caudate and 33% in the ventral striatum and olfactory tubercle labeled by the aFGF cRNA. These data demonstrate substantial regionally specific patterns of colocalization and support the hypothesis that, via an autocrine mechanism, aFGF provides local trophic support for cholinergic neurons in the basal forebrain and the striatum.

2213 Widespread co-localization of preprosomatostatin mRNA and β- preprotachykinin mRNA in the rat suprachiasmatic nucleus

2213 Widespread co-localization of preprosomatostatin mRNA and β- preprotachykinin mRNA in the rat suprachiasmatic nucleus

Colocalization of mRNA and protein using in situ hybridization and immunohistochemistry in testicular tissue.

Using testes fixed by perfusion with Bouin's fluid and embedded in paraffin wax, this study has established methods for combining in situ hybridization and immunocytochemistry on the same section to colocalize mRNA and protein for transition protein-1 (TP-1) and sulfated glycoprotein-1 (SGP-1), respectively. It was found that SGP-1 could be detected in tissue sections subsequent to the detection of TP-1 mRNA in situ. The finding that 1) the tissue pretreatments required to permeabilize the section and to allow access to the probe, and 2) the hybridization conditions themselves, had no adverse effect on the detection of antigen, eases the performance of this technique. On this basis, important information could be obtained on the transcriptional and translational activity of spermatogenic cells, if related probes and antibodies are utilized.

Anti-mullerian hormone and anti-mullerian hormone type II receptor messenger ribonucleic acid expression in rat ovaries during postnatal development, the estrous cycle, and gonadotropin-induced follicle growth

During fetal development, anti-müllerian hormone (AMH) is produced only by Sertoli cells, but postnatally, granulosa cells also produce this peptide growth/differentiation factor. We recently identified a candidate AMH type II receptor (AMHRII). In the present study, postnatal ovarian AMH and AMHRII messenger RNA (mRNA) expression was studied by in situ hybridization and ribonuclease protection. In ovaries from adult rats, AMH and AMHRII mRNAs were found to be mainly expressed in granulosa cells from preantral and small antral follicles. Corpora lutea and large antral follicles express little or no AMH and AMHRII mRNA, and primordial follicles and oocytes appeared to be AMH and AMHRII mRNA negative. Thecal and interstitial cells express no detectable AMH mRNA and little or no AMHRII mRNA. The colocalization of AMH and AMHRII mRNAs in granulosa cells of specific follicle types suggests that actions of AMH via AMHRII are autocrine in nature. There is a decreased level of AMH and AMHRII mRNA expression when follicles become atretic. Both mRNA species are eventually lost from atretic follicles, although AMHRII mRNA expression seems to persist somewhat longer than AMH mRNA. During the estrous cycle, no marked changes in the patterns of AMH and AMHRII mRNA expression were detected, except at estrus, when expression of both mRNA species in preantral follicles was decreased compared to that on the other days of the cycle. On postnatal day 5, total ovarian AMH mRNA expression is low and is located in small preantral follicles. During the first weeks of postnatal development, AMH mRNA expression in preantral follicles increases, and the later formed small antral follicles also express AMH mRNA. In contrast, AMHRII mRNA is expressed on postnatal day 5 at a higher level than AMH mRNA, but cannot be localized to specific cell types. From postnatal day 15 onward, AMHRII mRNA expression becomes more restricted to the preantral and small antral follicles. Treatment of prepubertal rats with GnRH antagonist (Org 30276) and human recombinant FSH (Org 32489) or with GnRH antagonist and estradiol benzoate resulted in follicle growth and inhibition of AMH and AMHRII mRNA expression in some, but not all, preantral and small antral follicles. These results indicate that FSH and estrogens may play a role in the down-regulation of AMH and AMHRII mRNA expression in vivo when small antral follicles differentiate into large antral follicles. Furthermore, the FSH surge on the morning of estrus may inhibit AMH and AMHRII mRNA expression in preantral follicles.