Studies on the development of growth hormone and prolactin cells in the rat pituitary gland by in situ hybridization

Summary An antiserum raised against N-amino-3-propyl melatonin bound to a protein carrier was used to visualize melatonin by immunohistochemistry and to measure melatonin concentration by radioimmunoassay in the pineal gland of intact mink females killed throughout the 24 h cycle and females killed after a bilateral ablation of the cervical superior ganglion. Melatonin immunoreactivity revealed by immunofluorescence or by the peroxidase-antiperoxidase complex was observed in the cytoplasm of presumed pinealocytes of all the females. Circadian changes in pineal melatonin content were not visualized by immunohistochemistry; furthermore, immunoreactivity was also present in the pineal gland of the ganglionectomized females. However, the melatonin content measured by radioimmunoassay was significantly higher in the pineal gland from intact females killed during the night compared with that of intact females killed during the day or of ganglionectomized females. The discrepancy between the results obtained using the two methods may arise because immunohistochemistry can detect very small amounts of melatonin.     

Ultrastructural distribution of growth hormone and prolactin mRNAs in normal rat pituitary cells: a comparison between preembedding and postembedding methods

In situ hybridization (ISH) at the electron microscopic level is essential for elucidating the intracellular distribution and role of mRNA in protein synthesis. We describe our electron microscopic ISH method using biotinylated oligonucleotide probes for rat growth hormone and prolactin mRNAs and compare the preembedding method with the postembedding method. Preembedding electron microscopic ISH localized rat growth hormone and prolactin mRNAs on the polysomes of the rough endoplasmic reticulum (RER). Rat growth hormone mRNA was distributed diffusely on the RER, whereas rat prolactin mRNA was scattered and distributed focally. Thus there might be a specific translational site for prolactin mRNA on the RER. Rat growth hormone mRNA signals were also recognized on the polysomes of the RER, using the postembedding method with streptavidin gold conjugate. The hybridization signal intensity using the postembedding method was lower, and non-specific signals were more frequent, in comparison with the preembedding method. The preembedding method thus appears to be easier and better than the postembedding method from the viewpoint of utility and preservation of mRNA. Electron microscopic ISH is considered to be an important tool for evaluating the intracellular localization of mRNA and the site of specific hormone synthesis on the RER.     

Fine-structural heterogeneity and morphologic changes in rat pituitary prolactin cells after estrogen and testosterone treatment

Summary This study was conducted to determine the functional and/or developmental relationships among three heterogeneous types of prolactin cells (I, II and III) in rats. Rats were injected subcutaneously daily with estradiol or testosterone propionate on days 10–20 after birth. Estradiol increased the proportion of cell types II and III, increased serum PRL levels 12-fold in males and 15-fold in females, and increased pituitary levels of prolactin 12-fold in males and 5-fold in females. Testosterone mainly increased the proportion of the Type-II cells, decreased serum levels of prolactin in males only, and did not change pituitary levels of prolactin. In a second experiment, treatment of rats with nafoxidine for five days after E₂ treatment (days 10–20 after birth) increased the proportion of Type-I cells and decreased the proportion of Type-III cells and decreased serum and pituitary levels of prolactin by 50% in females and by 15 and 45% in males. In a third experiment utilizing adult male rats, estradiol and testosterone were found to modulate the relative ratios of the different types of PRL cells as they did in immature animals. The data taken as a whole suggest the possibility of an estrogen-stimulated conversion of one cell type to another, which may be a reflection of prolactin secretory activity.This work was supported by a Scientific Research Grant from the Ministry of Education, Science and Culture of Japan (No. 57770038)     

Detection and localization of actin mRNA isoforms in chicken muscle cells by in situ hybridization using biotinated oligonucleotide probes

We have developed in situ hybridization methodology for nonisotopically labeled oligonucleotide probes to detect cellular mRNA with improved speed, convenience, and resolution over previous techniques. Previous work using isotopically labeled oligonucleotide probes characterized important parameters for in situ hybridization (Anal Biochem 166:389, 1987). Eleven oligonucleotide probes were made to coding and noncoding regions of chick beta-actin mRNA and one oligonucleotide probe to chick alpha-cardiac actin mRNA. All the probes were 3' end-labeled with bio-11-dUTP using terminal transferase, and the labeled probes were hybridized to chicken myoblast and myotube cultures. The hybridized probe was detected using a streptavidin-alkaline phosphatase conjugate. Our assay for the success of probe hybridization and detection was the demonstration of beta-actin mRNA highly localized in the lamellipodia of single cells (Lawrence and Singer, Cell 45:407, 1986) as well as the expression of alpha-cardiac actin mRNA and the repression of beta-actin mRNA in differentiating myoblasts and in myotubes. With the alpha-cardiac probe, we found that this mRNA was distributed all over the cytoplasm of myotubes and differentiated (bipolar) single cells and negative in undifferentiated single cells and at the ends of myotubes. When beta-actin probes were used, two of 11 probes were highly sensitive, and, in pooling them together, the localization of beta-actin mRNA in fibroblastic single cells was evident at the leading edge of the motile cells, the lamellipodium. beta-Actin mRNA was not detected in myotubes except at the ends where contact was made with substrate. This indicates that both beta and cardiac actin mRNA can coexist in the same myotube cytoplasm but at different locations.     

Co-localization of LH beta and FSH beta mRNAS in the porcine anterior pituitary by in situ hybridization with biotinylated probes

The localizations of mRNAs encoding LH beta and FSH beta in porcine pituitary were investigated by in situ hybridization technique. Biotinylated porcine LH beta and FSH beta cDNA probes were used on frozen sections of paraformaldehyde-fixed pituitary specimens. Hybridizations to both mRNAs were observed specifically in cytoplasm with unstained nuclei. Furthermore, cells hybridized for LH beta mRNA were demonstrated to be identical to those for FSH beta mRNA. This study provided the first morphological evidence that both gonadotropin beta genes are expressed in the same cell.     

Detection of Y-specific repeat sequences in normal and variant human chromosomes using in situ hybridization with biotinylated probes

In situ hybridization with a cloned human Y-specific repeat, pY3.4, derived from the 3.4-kb HaeIII repetitive sequences, is useful in identifying Yq-autosome translocations. In this study nonradioactive procedures were also employed to detect the sites of hybridization. Using a biotinylated probe and either immunofluorescence or horseradish peroxidase reaction, the chromosomes of three probands and members of their families with probable Y-autosome translocations were examined. It was found that not all such translocations can be correctly diagnosed based on conventional banding analysis. The present data indicate the importance of chromosome-specific probes in studying chromosome rearrangements in man.     

High-temperature fluorescent in situ hybridization for detecting Escherichia coli in seawater samples, using rRNA-targeted oligonucleotide probes and flow cytometry

Fluorescence in situ hybridization (FISH) is a widely used method to detect environmental microorganisms. The standard protocol is typically conducted at a temperature of 46 degrees C and a hybridization time of 2 or 3 h, using the fluorescence signal intensity as the sole parameter to evaluate the performance of FISH. This paper reports our results for optimizing the conditions of FISH using rRNA-targeted oligonucleotide probes and flow cytometry and the application of these protocols to the detection of Escherichia coli in seawater spiked with E.coli culture. We obtained two types of optimized protocols for FISH, which showed rapid results with a hybridization time of less than 30 min, with performance equivalent to or better than the standard protocol in terms of the fluorescence signal intensity and the FISH hybridization efficiency (i.e., the percentage of hybridized cells giving satisfactory fluorescence intensity): (i) one-step FISH (hybridization is conducted at 60 to 75 degrees C for 30 min) and (ii) two-step FISH (pretreatment in a 90 degrees C water bath for 5 min and a hybridizing step at 50 to 55 degrees C for 15 to 20 min). We also found that satisfactory fluorescence signal intensity does not necessarily guarantee satisfactory hybridization efficiency and the tightness of the targeted population when analyzed with a flow cytometer. We subsequently successfully applied the optimized protocols to E. coli-spiked seawater samples, i.e., obtained flow cytometric signatures where the E. coli population was well separated from other particles carrying fluorescence from nonspecific binding to probes or from autofluorescence, and had a good recovery rate of the spiked E. coli cells (90%).     

Bioactivity of plasma prolactin in ovariectomized, diethylstilbestrol-treated Long-Evans and Holtzman rats after thyrotropin-releasing hormone or bromocriptine administration

The objective of this study was to determine the effects of thyrotropin-releasing hormone (TRH) and bromocriptine on plasma levels of biologically active prolactin in ovariectomized, diethylstilbestrol (DES)-treated rats. Female Long-Evans and Holtzman rats were ovariectomized and each was given a subcutaneous implant of diethylstilbestrol (DES). One week later, groups of DES-treated rats were fitted with indwelling intra-atrial catheters, and 2 days later blood samples were withdrawn before and at 1, 2, 5, 10, and 20 min after intravenous administration of TRH (250, 500, or 1000 ng/rat). Blood samples were obtained from other groups at 4 weeks of DES treatment by orbital sinus puncture under ether anesthesia before and at 30, 60, and 120 min after bromocriptine administration (2.5 mg/rat sc). Plasma was assayed for prolactin by conventional radioimmunoassay (RIA) and by Nb2 lymphoma bioassay (BA). Holtzman rats released significantly more prolactin following TRH than did Long-Evans rats when the RIA was used to measure prolactin. However, when the BA was used to assay prolactin in the same samples, the Long-Evans rats released more prolactin than did the Holtzman rats. In addition, the ratio of the BA to RIA values was significantly increased in both strains following TRH, but the greatest increase was observed in the Long-Evans rats, in which the ratio was 4.5 at the peak of the TRH-induced rise in plasma prolactin. Gel filtration chromatography of plasma obtained at 5 min after TRH treatment in Long-Evans rats revealed large molecular forms of prolactin with BA to RIA ratios of 4-5. In addition, monomeric prolactin had a BA to RIA ratio of 2. Bromocriptine treatment reduced prolactin levels in both strains, but the effect was more rapid in Holtzman than in Long-Evans rats. In addition, bromocriptine treatment of Holtzman, but not Long-Evans, rats significantly reduced the BA to RIA ratio of plasma prolactin. The results indicate that TRH and bromocriptine affect the release of biologically active prolactin to a greater extent than prolactin detected by antibody in the RIA, and that Long-Evans and Holtzman rats respond to these secretagogues differently with regard to BA to RIA comparisons.     

Differential localization of mRNAs of collagen types I and II in chick fibroblasts, chondrocytes, and corneal cells by in situ hybridization using cDNA probes

We have employed a highly specific in situ hybridization protocol that allows differential detection of mRNAs of collagen types I and II in paraffin sections from chick embryo tissues. All probes were cDNA restriction fragments encoding portions of the C-propeptide region of the pro alpha-chain, and some of the fragments also encoded the 3'-untranslated region of mRNAs of either type I or type II collagen. Smears of tendon fibroblasts and those of sternal chondrocytes from 17-d-old chick embryos as well as paraffin sections of 10-d-old whole embryos and of the cornea of 6.5-d-old embryos were hybridized with 3H-labeled probes for either type I or type II collagen mRNA. Autoradiographs revealed that the labeling was prominent in tendon fibroblasts with the type I collagen probe and in sternal chondrocytes with the type II collagen probe; that in the cartilage of sclera and limbs from 10-d-old embryos, the type I probe showed strong labeling of fibroblast sheets surrounding the cartilage and of a few chondrocytes in the cartilage, whereas the type II probe labeled chondrocytes intensely and only a few fibroblasts; and that in the cornea of 6.5-d-old embryos, the type I probe labeled the epithelial cells and fibroblasts in the stroma heavily, and the endothelial cells slightly, whereas the type II probe labeled almost exclusively the epithelial cells except for a slight labeling in the endothelial cells. These data indicate that embryonic tissues express these two collagen genes separately and/or simultaneously and offer new approaches to the study of the cellular regulation of extracellular matrix components.     

Albumin and collagen mRNA expression in normal and analbuminemic rodent liver: analysis by in situ hybridization using biotinylated probes

We used in situ nucleic acid hybridization cytochemistry to examine cell types and subcellular sites expressing albumin (alb) or pro alpha 2 collagen (col) mRNA in livers from normal and analbuminemic rodents. Biotinylated cDNA or RNA probes were applied to aldehyde-fixed, non-frozen sections and the resulting DNA-RNA or RNA-RNA hybrids were subsequently visualized by enzymatic detection of either peroxidase or alkaline phosphatase conjugated to anti-biotin IgG or streptavidin. In normal rat liver, alb mRNA was expressed in all hepatocytes and was localized to discrete subcellular structures distributed as aggregates in the cytoplasm and in specific structures encircling the nucleus; these subcellular structures most likely represent the endoplasmic reticulum and nuclear envelope. In mouse liver, pro alpha 2 col mRNA was identified in a subpopulation of sinusoidal lining cells which have the morphological appearance of lipocytes. In liver from analbuminemic rats, a small number of hepatocytes, distributed throughout the hepatic lobule, expressed alb mRNA at high levels; the subcellular distribution of this alb mRNA was essentially identical to that observed in normal rat hepatocytes. Since non-radioactive in situ hybridization detected mRNA within the boundaries of individual cells and showed its precise subcellular location under conditions in which there was excellent preservation of tissue morphology, this procedure should be useful for a wide variety of histopathologic studies.     

Estrogen stimulates both prolactin and growth hormone mRNAs expression in the MtT/F4 transplantable pituitary tumor

The effects of 17 beta-estradiol (E2) on MtT/F4 pituitary tumor growth and on prolactin (PRL) and growth hormone mRNA expression were analyzed in F344 female rats. E2 (10 mg) stimulated pituitary PRL cell hyperplasia and PRL mRNA, but inhibited growth of the transplantable tumors. The expression of both PRL and growth hormone mRNA levels was increased in the MtT/F4 tumors. The effects of E2 on increasing PRL mRNA levels were more marked in the pituitary compared with the tumors. These results indicate that estrogens stimulate proliferation and PRL expression in the pituitary while inhibiting cell proliferation in the MtT/F4 tumor. E2 also stimulated both growth hormone and PRL mRNA expression in the MtT/F4 transplantable tumor.     

Effect of estradiol 17-beta on LH subunits and prolactin mRNAs expression in the pituitary of old female rats

OBJECTIVES: The aim of the study was to examine susceptibility of the pituitary gland to estrogenic impulse in old, noncycling rats by measurement of steady state level of mRNAs encoding LH subunits a and b and mRNA for PRL. METHODS: 22-month-old rats were ovariectomized and after one week they were subcutaneously implanted with silastic tubing filled with oil or with estradiol 17-beta. Pituitary alpha, LHbeta and PRL mRNAs content and serum LH and PRL concentration was determined. RESULTS: The effect of E (2)treatment was manifested by the significant increase in the weight of the uterus and pituitary gland as well as by elevation of total pituitary RNA (109%, 60% and 78%, respectively; p<0.001). No significant changes (p>0.05) in serum LH concentration were observed, while levels of mRNAs encoding alpha and LH-beta subunits were lowered by 54% (p<0.05) and 96% (p<0.01), respectively, in the rats subjected to E(2) stimuli. No direct correlation between synthesis and release of LH in E(2) treated old rats was observed. The blood PRL concentration and the pituitary level of PRL mRNA increased up to 2,000% and 1,300%, respectively (p<0.001). Spontaneous pituitary adenoma was observed in about 30% of the rats, irrespective of treatment. CONCLUSIONS: These data show that in old rats estrogenic stimulus can effectively diminish both pituitary LH subunits mRNAs as well as stimulate pituitary PRL mRNA level indicating that the E(2)-dependent processes involved in the regulation of corresponding genes are still functional.     

Subcellular distribution of laminin and prolactin in stimulated and blocked prolactin cells in the pituitary of lactating rats

Summary Laminin (LAM), a glycoprotein component of basement membranes, has been previously detected within several subcellular compartments of prolactin (PRL) cells in the pituitary gland. The present work was aimed at comparing the subcellular localization of PRL, a specific secretory product, with that of LAM, in relation to the secretory activity of PRL cells. LAM and PRL were located in parallel, by ultrastructural immunocytochemistry, in PRL cells of lactating female Wistar rats, either stimulated by suckling, or blocked by weaning, or reactivated by suckle following short-term weaning. Variations in physiological conditions were correlated with a redistribution of PRL immunoreactivity within morphologically modified compartments. The Golgi apparatus became hypertrophied, and PRL impressively accumulated within saccules of the Golgi stacks of blocked cells. On the contrary, no apparent changes occurred in LAM distribution, at least at the Golgi level. Only a slight increase of LAM immunoreactivity was observed in rough endoplasmic reticulum after a long weaning period. PRL could be detected in most of the secretory granules and particularly in forming elements, whereas LAM was observable at the peripheral edge of some mature granules. Such a labeling was not markedly influenced by the physiological state. The prominent structures, indicative of crinophagic activity, characteristic of blocked cells, contained masses of dense material, which were always immunopositive with antibodies to PRL, but never to LAM. These observations could suggest that, in PRL cells, intracellular transport and exportation of LAM are controlled by mechanisms independent from those involved in the regulation of PRL secretion.     

Cell cycle distributions, growth characteristics, and variation in prolactin and growth hormone production in cultured rat pituitary cells.

Clonal strains of rat pituitary tumour cells (GH3 cells) spontaneously produce and secrete prolactin and growth hormone. Chromosome analysis and DNA ploidy measurements revealed that the GH3 cells in the present study were triploid and had a decreased chromosome number compared to the parent strain. Monolayer cultures of these cells grow exponentially for 6-7 days with a mean doubling time of 54 h. Cell cycle distributions and phase durations were determined by micro-flow fluorometric measurements of cellular DNA content combined with computer calculations. During exponential growth the cell cycle distribution did not change (65.4% cells with a G1 phase DNA content, 24.9% with an S phase DNA content, and 9.7% with a (G2 + M) phase DNA content). Counting of mitoses gave 1.4% cells in M phase. The 3H-Tdr labeling indices were determined by autoradiography, and the results were in good agreement with the number of cells in S phase as calculated by micro-flow fluorometry. The phase durations were: Ts=15.9 h, TG2=6.2 h, TM=1.1 h, and TG1=30.9 h. TS and TM calculated from 3H-Tdr labeled and Colcemid treated cultures gave corresponding results. In plateau phase cultures the number of cells with a G1 DNA content increased to 80% and the number of cells with an S phase DNA content decreased to between 5% and 10%. The specific production of prolactin and growth hormone determined by radioimmunoassay showed two and four-fold increases respectively, during exponential growth. The hormone values decreased to initial or subinitial values (day 2 values) when approaching plateau phase. We conclude: that changes in the cell cycle distribution of the cell population cannot be responsible for the spontaneous alterations in hormone production during growth and that most of the hormone-producing cells must be in the G1 phase.