Estrogen induced prolactin mRNA accumulation in adult male rat pituitary as revealed by in situ hybridization

Adult male rats were injected with different doses (1, 10, and 100 micrograms) of 17 beta-estradiol daily for 5 days, and the changes in prolactin (PRL) mRNA levels were examined by in situ hybridization and cytoplasmic dot blot hybridization using cloned cDNA for rat prolactin mRNA. An increase in cytoplasmic PRL mRNA content was evident in all the animals treated with estrogen as revealed with cytoplasmic dot blot analysis. There were however, no significant differences in PRL mRNA content among the three estradiol treated groups. Cytoplasmic PRL mRNA was also demonstrated by in situ hybridization on the frozen pituitary sections using a 3H-labeled PRL cDNA probe. The number of grains per cell was increased after estrogen treatment. 3H-thymidine uptake into pituitary cells was also examined in vivo using combined techniques of immunocytochemistry and autoradiography. Although the percentage of immunoreactive PRL cells which took up thymidine in their nuclei increased to more than double after estrogen treatment, the increase in the total number of immunoreactive PRL cells was small. These results suggest that the major effect of estrogen on PRL cells is an increase in the accumulation of PRL mRNA in the individual PRL cells. The number of grains per cell was found to vary from cell to cell, both in control and estrogen treated animals. This variability is discussed in relation to the functional heterogeneity within the PRL cell population.     

Ultrastructural non-radioactive in situ hybridization of GH mRNA in rat pituitary gland: pre-embedding vs ultra-thin frozen sections vs post-embedding.

In situ hybridization at the ultrastructural level can be carried out using three different methods: on vibratome sections before embedding in epoxy resin, on ultra-thin frozen sections, or on ultra-thin sections of tissues embedded in hydrophilic resin such as Lowicryl. With the purpose of comparing the sensitivity, resolution, and ultrastructural preservation of these three methods, we examined the expression of the growth hormone (GH) gene in anterior pituitary cells by in situ hybridization at the ultrastructural level, using a synthetic oligonucleotide complementary to the codons of the mRNA from Gln 45 to Ser 54 labeled at the 3' end of biotin-21dUTP. All these methods gave similar results: mRNA was located on the lamellar endoplasmic reticulum of somatotrophs. The pre-embedding method gave the best ultrastructural preservation, with low resolution with the enzymatic detection system and an intermediate sensitivity. A probe concentration of 10 pmol/ml was sufficient to obtain a signal. With this method gold particles could not be used without pre-treatment. The frozen section method gave the best sensitivity (a signal was observed with 4 pmol/ml of probe) but the lowest ultrastructural preservation. On ultra-thin Lowicryl sections, resolution was as high as with the frozen-section method, ultrastructural conservation was intermediate, and sensitivity was low. These results indicate that the last method seems to be a good compromise between sensitivity and ultrastructural preservation.     

Immunocytochemical study of the GH cells in the anterior pituitary gland of human fetus II. Anencephalic fetus

In order to elucidate the effects of hypothalamic regulation on the morphology of GH cells, light and electron microscopic immunocytochemical examinations were carried out comparing GH cells in the anterior pituitary gland of anencephalic fetus with those of normal fetuses. Three types of GH cells were identified in the anterior pituitary gland of anencephalic fetus as well as in the normal fetus. Type-I is a small, round cell containing a few small secretory granules. Type-III is a large, polygonal cell with numerous large secretory granules. Type-II is a polygonal cell with medium-sized secretory granules. The Type-II GH cell was predominant in both anencephalic and normal fetuses. The most striking difference between anencephalic and normal fetuses was the presence of atypical forms of the Type II cell. These were polygonal cells containing secretory granules, which were either immunopositive or immunonegative to anti-human GH (anti-hGH) serum. Furthermore, two other types of GH cells were identified. The somatomammotroph (SM cell) contained GH and PRL in different granules within the same cell. Also, a different type of the GH cell was noted containing two varieties of secretory granules; one was immunolabeled only with anti-hGH and the other was not immunolabeled to either anti-hGH or anti-human PRL (anti-hPRL). From these results, we suggest that an absence of hypothalamic regulation in the anencehpalic does not seriously modify GH cell morphology but induces an altered GH storage pattern in some of the cells.     

Discrepancy between prolactin (PRL) messenger ribonucleic acid and PRL content in rat fetal pituitary cells: possible role of dopamine

Data are controversial concerning the time when PRL-synthesizing cells are detected for the first time in the rat pituitary. Using a very sensitive immunocytochemical technique, we could visualize only a few PRL cells before day 10 after birth. At that time, pituitary PRL was still 200 times less abundant than in the adult (on a tissue weight basis) whereas PRL mRNA per mg total RNA was only 80 times lower than in the adult. However, by in situ hybridization, we could demonstrate the presence of PRL mRNA in cells from fetal day 18 on. We have also followed the expression of GH gene in rat pituitary cells during development. In contrast to results obtained with PRL cells, quantitative analysis of cDNA probe hybridization to GH mRNA correlated well with measurements of immunostained cells. We found that PRL was released in the blood from fetal day 19 onwards. Thus, at that time PRL is synthesized and secreted but not stored. We therefore measured brain dopamine levels, and the data support the idea that the rise in dopamine levels after birth contributes to PRL storage. We confirmed in vitro that newborn pituitary cells can store PRL when cultured in the presence of dopamine.     

Improved detection of in situ hybridization by laser scanning confocal microscopy.

Laser scanning confocal microscopy (LSCM) offers a significant improvement over conventional bright-field and dark-field light microscopy for producing images of silver grains in autoradiograms of specimens prepared by in situ hybridization. The out-of-focus image of the background silver grains present in the emulsion is eliminated from the in-focus image of the radioactive probe associated with the cells by optical sectioning with the LSCM operated in a reflected light mode. The improved images produced by the LSCM provide a significant increase in the sensitivity of detecting positively labeled cells and tissues prepared by in situ hybridization. The power of this detection method is demonstrated using samples of HIV-infected human peripheral blood cells, tissue sections of human placenta and human skin. It is anticipated that the method can be universally applied to samples prepared by in situ hybridization techniques.     

Localization of nerve growth factor receptor mRNA in the rat basal forebrain within situ hybridization histochemistry

1. In situ hybridization histochemistry was used to localize nerve growth factor receptor (NGFR) mRNA in the adult rat basal forebrain. 2. In emulsion-dipped sections 35S-labeled RNA antisense probes produced a high density of silver grains over cells located in the medial septum, vertical and horizontal limbs of the diagonal band of Broca, and nucleus basalis. 3. This distribution of NGFR mRNA overlaps with the distribution of NGFR protein localized using immunocytochemical techniques. 4. No hybridization signal was detected when sections were hybridized with a 35S-labeled RNA sense (control) probe. 5. We suggest that NGFRs are synthesized in these basal forebrain nuclei and transported to terminal areas where NGF is thought to be bound and internalized, an initial step in the many actions of this neurotrophic factor.     

Complementary distribution of carbamoylphosphate synthetase (ammonia) and glutamine synthetase in rat liver acinus is regulated at a pretranslational level

We studied the distribution of the mRNAs for carbamoylphosphate synthetase (ammonia) and glutamine synthetase in frozen sections of adult rat liver by in situ hybridization to [35S]-labeled cDNA probes. The density of silver grains resulting from hybridization to the labeled cDNA probe for carbamoylphosphate synthetase is highest around the portal venules, decreases towards the central venule, and is virtually absent from an area two to three cells wide that lines the central venules in which mRNA for glutamine synthetase is predominantly localized. Therefore, both mRNAs show the same complementary distribution within the liver acinus that was found for the proteins they encode, demonstrating that compartmentalization of the expression of these enzymes is controlled at a pretranslational level. In addition, we found that carbamoylphosphate synthetase mRNA is present mainly in the epithelium of the crypts of the proximal part of the small intestine, whereas carbamoylphosphate synthetase protein is present in the epithelium of both crypts and villi.     

Subcellular distribution of secretogranins I and II in GH3 rat tumoral prolactin (PRL) cells as revealed by electron microscopic immunocytochemistry

The GH3 rat pituitary cell line which secretes prolactin (PRL) is characterized by the paucity and small size of secretory granules. We looked for the presence, in these cells and in normal PRL cells, of two acidic tyrosine-sulfated proteins which are widely distributed in dense-core secretory granules of endocrine and neuronal cells, secretogranins I and II, using immunofluorescence and electron microscope immunoperoxidase techniques. Both secretogranins were detected in secretory granules of GH3 cells and of normal cells. Moreover, with our pre-embedding approach, secretogranins were localized within some RER cisternae and within all sacules of the Golgi stacks in both PRL cell models. A few small vesicles, large dilated vacuolar or multivesicular structures, and some lysosome-like structures were also immunoreactive. Double localization of secretogranins and PRL performed on GH3 cells by immunofluorescence indicated that all cells contained secretogranins I and II, whereas only 50-70% of the cells contained PRL. Moreover, in the case of hormone treatment known to increase the number of secretory granules, most if not all mature secretory granules were immunoreactive for secretogranins, whereas in certain cells some of the granules were apparently not immunoreactive for PRL. These immunocytochemical observations show that GH3 cells, which under normal conditions form only a small number of secretory granules, produce secretogranins and package them into these granules.     

Chromogranin A biosynthetic cell populations in bovine endocrine and neuronal tissues: detection by in situ hybridization histochemistry

Chromogranin A is a highly acidic protein that is found in the secretory granules of many endocrine and neuronal cells. To localize bovine cell populations involved in chromogranin A biosynthesis, the distribution of the mRNA encoding this protein was determined with in situ hybridization histochemistry. In the adrenal gland, the mRNA was found in the chromaffin cells of the medulla but was absent from the cortex. The distribution of the mRNA in the medulla was uneven; cells located at the periphery were more heavily labeled than those in the center of the gland. Because the adrenal medulla is composed of several cell types, the chromogranin A-containing cells were further characterized for the presence of neuropeptide and adrenergic markers. Adjacent sections were examined for the mRNAs encoding enkephalin and phenylethanolamine N-methyltransferase (PNMT), the enzyme that catalyzes the formation of epinephrine from norepinephrine. Both mRNAs were present in a narrow band of cells at the periphery of the medulla. However, in contrast to the distribution of chromogranin A mRNA, the enkephalin and PNMT mRNAs were detected in only a small number of cells in the inner medullary region. The difference in the distribution of the enkephalin and PNMT mRNAs from that of chromogranin A suggests that the expression of these genes is differentially regulated. In addition to the adrenal gland, chromogranin A mRNA is expressed by many other tissues. In the parathyroid gland, which is rich in the mRNA but exhibits little chromogranin A-like immunoreactivity, the message was present in most cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electron microscopic detection of RNA sequences by non-radioactive in situ hybridization in the mollusk Lymnaea stagnalis.

The subcellular localization of mRNA sequences encoding neuropeptides in neuropeptidergic cells of the pond snail Lymnaea stagnalis was investigated at the electron microscopic (EM) level by non-radioactive in situ hybridization. Various classes of probes specific for 28S rRNA and for the ovulation hormone (caudodorsal cell hormone; CDCH) mRNA were labeled with biotin or digoxigenin and were detected after hybridization with gold-labeled antibodies. Hybridizations were performed on ultra-thin sections of both Lowicryl-embedded and frozen cerebral ganglia, and a comparison demonstrated that most intense hybridization signals with an acceptable preservation of morphology were obtained with ultra-thin cryosections. Addition of 0.1% glutaraldehyde to the formaldehyde fixative improved the morphology, but on Lowicryl sections this added fixative resulted in a decrease of label intensity. A variety of probes, including plasmids, PCR products, and oligonucleotides, were used and all provided good results, although the use of oligonucleotides on Lowicryl sections resulted in decreased gold labeling. The gold particles were found mainly associated with rough endoplasmic reticulum (RER) but were also observed in lysosomal structures. Finally, the in situ hybridization method presented in this study proved to be compatible with the immunocytochemical detection of the caudodorsal cell hormone, as demonstrated by double labeling experiments.     

Immunocytochemical and ultrastructural characterization of mammosomatotrope-, growth hormone-, and prolactin-cells from the gilthead sea bream (Sparus aurata l., Teleostei): an ontogenic study

Growth hormone (GH), prolactin (PRL), and mammosomatotrope (MS) cells of gilthead sea bream, Sparus aurata, a teleost fish, were studied in specimens from hatching to 15 months (adults) using conventional electron microscopy and an immunogold method using anti-tilapia GH sera and anti-chum salmon PRL serum. MS cells, immunoreactive to both anti-GH sera and anti-PRL sera, had been first identified in fish in a previous study in newly hatched larvae and in older larvae and juvenile specimens of Sparus aurata by light microscopic immunocytochemistry. In the present work, MS cells reacted positively to immunogold label only in older larvae and juveniles and their secretory granules immunoreacted with both GH and PRL antisera or with only one of them. MS cells were ultrastructurally similar to the PRL cells, with which they coincided in time. This is the first report on the ultrastructural characterization of MS cells in fish. In adults, the secretory granules of GH cells (immunoreactive to anti-GH serum) were mainly round, of variable size, and had a homogeneous, highly electron-dense content. Irregularly shaped secretory granules were also present. PRL cells (immunoreactive to anti-PRL serum) were usually observed in a follicular arrangement; they showed few, small, and mainly round secretory granules with a homogeneous and high or medium electron-dense content. Some oval or elongated secretory granules were also observed. GH and PRL cells that showed involutive features were also found. In newly hatched larvae, GH, PRL, and MS cells could not be distinguished either by their ultrastructure or by the immunogold labeling of the secretory granules. In 1-day-old larvae, presumptive GH and PRL cells were observed according to their position in the pituitary gland. In 2-day-old larvae, a few cells showed some of the ultrastructural features described for GH and PRL cells of adults. During development, the number, size, and shape of the secretory granules in both cell types clearly increased and the organelles developed gradually. Some GH cells were found undergoing mitosis.     

Sorting of three secretory proteins to distinct secretory granules in acidophilic cells of cow anterior pituitary

The distribution of three proteins discharged by regulated exocytosis--growth hormone (GH), prolactin (PRL), and secretogranin II (SgII)--was investigated by double immunolabeling of ultrathin frozen sections in the acidophilic cells of the bovine pituitary. In mammotrophs, heavy PRL labeling was observed over secretory granule matrices (including the immature matrices at the trans Golgi surface) and also over Golgi cisternae. In contrast, in somatotrophs heavy GH labeling was restricted to the granule matrices; vesicles and tubules at the trans Golgi region showed some and the Golgi cisternae only sparse labeling. All somatotrophs and mammotrophs were heavily positive for GH and PRL, respectively, and were found to contain small amounts of the other hormone as well, which, however, was almost completely absent from granules, and was more concentrated in the Golgi complex, admixed with the predominant hormone. Mixed somatomammotrophs (approximately 26% of the acidophilic cells) were heavily positive for both GH and PRL. Although admixed within Golgi cisternae, the two hormones were stored separately within distinct granule types. A third type of granule was found to contain SgII. Spillage of small amounts of each of the three secretory proteins into granules containing predominantly another protein was common, but true intermixing (i.e., coexistence within single granules of comparable amounts of two proteins) was very rare. It is concluded that in the regulated pathway of acidophilic pituitary, cell mechanisms exist that cause sorting of the three secretory proteins investigated. Such mechanisms operate beyond the Golgi cisternae, possibly at the sites where condensation of secretion products into granule matrices takes place.     

Effects of light deprivation on prolactin cells in golden hamsters: an immunoelectron microscopic study.

In the golden hamster light deprivation has been shown to induce gonadal regression and reduction of pituitary and plasma levels of prolactin (PRL). In the present study we examined changes in morphology and population ratios of three types of PRL cells 8 weeks after light deprivation, by means of blinding or exposure of hamsters to continuous darkness. In the pituitary of intact hamsters of either sex, which were entrained to a 14-h light: 10-h dark cycle, Type C cells with large secretory granules were the most numerous and Type A with smaller granules the least. After light deprivation the pituitary was found to contain remarkably atrophic PRL cells and showed a profound change in population ratio of PRL cell types, i.e., Type A cells prevailed over the other two types. Pituitary glands from light-deprived and concurrently pinealectomized hamsters exhibited structures and a population ratio of three types of PRL cells similar to those from intact animals. It is suggested that small-granule-containing PRL cells represent an inactive stage of PRL cells, whereas medium- and large-granule-containing cells are functionally active cells. The atrophy of PRL cells can account for the decreased pituitary level of PRL in light-deprived hamsters reported previously.     

Nonradioactive in Situ Hybridization Histochemistry in Leukemic and Nonleukemic Culture

Technical limitations are associated with conducting successful in situ hybridization. In this study, three cell types including a tumor neuroblastoma cell line (Neuro-2a), an oligodendrocyte primary culture, and a nonneuronal acute lymphoblastic leukemia cell line (Reh) were used to conduct successful nonradioactive in situ hybridization. Two cDNA probes were used. A 1 kb probe was used to identify the expression of proteolipid protein (PLP) mRNA in a primary culture of oligodendrocytes. A 760 bp cDNA was used to identify the expression of ubiquitin C-terminal hydrolase (UCH-L1) mRNA in Neuro-2a and Reh cells. The probes were labeled with digoxigenin-11-dUTP, denatured, and hybridized with cells fixed on coverslips. The efficiency of the labeling was tested using dot blot analysis by comparing the intensity of our labeled probes with known concentration of the probe labeled by the provider. The nonspecific signals were washed off, followed by detection of a signal specific to the gene. The specificity of the probes was determined by treating the cells with RNase A, hybridizing with bacterial Dig-labeled cDNA (pBR322) and hybridizing the tissues in the absence of labeled probe. During the labeling step, we found that addition of co-precipitants, such as tRNA or glycogen, during precipitation of the labeled probe followed by overnight incubation at -20 C is essential for good recovery of labeled cDNA. Dissolving the labeled probe in a buffer solution containing sodium dodecyl sulfate improves the quantity of the labeling. At the cellular level, prehybridization treatments optimize the permeability of the cell and allow efficient penetration of the labeled probe. Fixing with paraformaldehyde or an ethanol-acetic acid mixture can preserve the structure of cultured cells. To increase the signal to noise ratio, cells were treated with 0.2 N HC1 followed by extensive washes using a solution with a high salt concentration and containing dextran sulfate. This treatment significantly improves the signal and reduces the background in cell cultures, but not in tissue sections. The ability to reuse the labeled probe-hybridization mixture is another advantage for using nonradioactive in situ hybridization.     

Incorporation of L-(3H)-fucose into glycoproteins of the adrenal gland of mice. Light microscope radioautographic study on semi-thin sections

Summary To study the biosynthesis and intracellular migration of glycoproteins in the adrenal gland, adult mice were injected intravenously with L-(³H) fucose and killed from 10 min to 14 days after injection. Semi-thin sections of the adrenal glands were then processed for radioautography. Incorporation of labeled fucose occurred in the steroid-secreting cells of the three zones of the cortex as well as in the adrenalin (A) and noradrenalin (NA) cells of the medulla. At short intervals after injection, the main site of incorporation was the paranuclear region of the cells, suggesting uptake by the Golgi apparatus. Subsequently, labeled glycoproteins migrated from the paranuclear region to other cell sites. The labeling pattern observed in the adrenocortical parenchyme strongly suggests that the glycoproteins are transferred to lysosomes, lipofuscin granules and the cell coat (glycocalyx). Counts of silver grains clearly indicate that these glycoproteins undergo renewal. The qualitative and quantitative analysis of the radioautographs also suggest that glycoproteins, acting as intracellular carriers of steroids, may be released to the extracellular environment together with the hormones. Most of the glycoproteins synthesized by the A and NA cells of the adrenal medulla seem to be transferred to secretion granules in which they may play some role in the cytophysiology of these structures. It is likely that glycoproteins are released from the cells during exocytosis of secretory granules.     

Ultrastructural localization of fibronectin mRNA in chick embryo by in situ hybridization using 35S or biotin labeled cDNA probes

We have studied the expression of the fibronectin gene in 7 day-old chick embryo (stage 32) by in situ hybridization at the light and electron microscope levels, using a 397 base-pairs chicken cDNA, labeled by radioisotope or biotin-11dUTP. Cryostat sections of whole chick embryos displayed a selective label on the upper layer of the dermis, fibrous sclera and mesenchymal cells but not on cartilagenous sclera cells. These results show that the expression of the fibronectin gene varies in relation to the morphogenetic events. Hybridization at the ultrastructural level on thin sections of sclera embedded in Lowicryl K4M showed a selective labelling on various cell compartments. Biotin-11dUTP and radiolabeled probes were compared. The labeling was found precisely on the membrane of the rough endoplasmic reticulum and on the nuclear envelope. A few silver grains were located on the nucleus and in the perinucleolar region. This study shows that the postembedding in situ hybridization is a powerful procedure to study the expression of the extracellular protein genes and gives further information on the localization of mRNA.