Changes in Laminin Chain Expression in Pre- and Postnatal Rat Pituitary Gland

Cell–matrix interaction is required for tissue development. Laminin, a major constituent of the basement membrane, is important for structural support and as a ligand in tissue development. Laminin has 19 isoforms, which are determined by combinational assembly of five α, three β, and three γ chains (eg, laminin 121 is α1, β2, and γ1). However, no report has identified the laminin isoforms expressed during pituitary development. We used in situ hybridization to investigate all laminin chains expressed during rat anterior pituitary development. The α5 chain was expressed during early pituitary development (embryonic day 12.5–15.5). Expression of α1 and α4 chains was noted in vasculature cells at embryonic day 19.5, but later diminished. The α1 chain was re-expressed in parenchymal cells of anterior lobe from postnatal day 10 (P10), while the α4 chain was present in vasculature cells from P30. The α2 and α3 chains were transiently expressed in vasculature cells and anterior lobe, respectively, only at P30. Widespread distribution of β and γ chains was also observed during development. These findings suggest that numerous laminin isoforms are involved in anterior pituitary gland development and that alteration of the expression pattern is required for proper development of the gland.     

Laminin Isoforms and Laminin-Producing Cells in Rat Anterior Pituitary

Laminin is a key component of the basement membrane and is involved in the structural scaffold and in cell proliferation and differentiation. Research has identified 19 laminin isoforms, which are assemblies of α, β, and γ chains (eg, the α1, β1, and γ1 chains form the laminin 111 isoform). Although laminin is known to be present in the anterior pituitary, the specific laminin isoforms have not been identified. This study used molecular biological and histochemical techniques—namely, RT-PCR, immunohistochemistry, and in situ hybridization—to identify the laminin isoforms and laminin-producing cells in rat anterior pituitary. RT-PCR showed that laminin α1, α3, and α4 genes were expressed in anterior pituitary. Immunohistochemistry revealed laminin α1 in gonadotrophs and laminin α4 in almost all vascular endothelial cells. Laminin α3 was seen in a subset of vascular endothelial cells. We then performed in situ hybridization to localize β and γ chains in these cells and found that laminin β1, β2, and γ1 were expressed in gonadotrophs and that laminin β1 and γ1 were expressed in endothelial cells. In conclusion, we identified gonadotroph-type (laminin 111 and 121) and vascular-type (laminin 411 and 311) laminin isoforms in rat anterior pituitary.     

Presence of prolactin mRNA in extra-pituitary brain areas in the domestic turkey

It is well known that prolactin plays diverse roles in vertebrate reproduction. Besides expression in the pituitary, prolactin is also found in extra-pituitary tissues. In the present study, prolactin mRNA expression was studied utilizing in situ hybridization histochemistry. Prolactin mRNA, while found throughout the turkey brain, was predominantly localized within the pituitary, confirming a pivotal role of prolactin in turkey reproduction. The expression of prolactin mRNA was also observed within extra-pituitary brain areas including the cerebellum, nucleus accumbens, lateral septum, anterior hypothalamic nucleus, lateral hypothalamus, paraventricular nucleus, ventromedial nucleus, and infundibular nuclear complex. In the hypothalamus, an abundance of prolactin mRNA-expressing cells was observed in the anterior hypothalamic nucleus, lateral hypothalamus, and ventromedial nucleus. Cells expressing the least prolactin mRNA were found in the lateral septum, paraventricular nucleus, and the infundibular nuclear complex. This study reveals, for the first time, that prolactin mRNA was expressed in extra-pituitary brain areas in birds. In addition, the diverse expression of prolactin mRNA in the brain areas suggests that prolactin plays various physiological roles in birds.     

The Herbal Medicine Unkei-To Stimulates Cytokine-Induced Neutrophil Chemoattractant Production in the Pituitary Folliculo-Stellate—Like Cell Line (TtT/GF)

PROBLEM: We previously reported that a cytokine-induced neutrophil chemoattractant (CINC) was produced in the pituitary gland and that it influenced anterior pituitary hormone release. In this study we investigated the effect of Unkei-to, a Japanese herbal medicine, on CINC production in the rat anterior pituitary gland and the pituitary folliculo-stellate-like cell line (TtT/GF). METHOD OF STUDY: Dispersed normal anterior pituitary cells and the folliculo-stellate-like cell line TtT/GF were used to test the effect of Unkei-to on CINC secretion and CINC mRNA accumulation. Concentrations of CINC in the conditioned media were measured by an enzyme-linked immunosorbent assay, and levels of CINC mRNA were analyzed by Northern blot analysis. RESULTS: Unkei-to (20 μg/ml) significantly increased the secretion of CINC by normal anterior pituitary cells within 12 hr of incubation. Unkei-to also stimulated CINC secretion from TtT/GF cells in a time- and dose-dependent manner. Unkei-to (20 μg/ml) increased CINC mRNA accumulation in TtT/GF cells within 3 hr of incubation and also caused a 13-fold increase in the secretion of CINC from TtT/GF cells compared with the vehicle group within 24 hr of incubation. Finally, we found that some of the Unkei-to's ingredients, Evodiae fructus and Pinelliae tuber, markedly stimulated CINC secretion from TtT/GF cells. CONCLUSIONS: Our results will help to elucidate the mechanism behind the clinical effect of Unkei-to on the anterior pituitary gland. They also suggested the presence of special substances, which stimulate CINC secretion, within Unkei-to's ingredients such as E. fructus and P. tuber.     

Detection of the messenger RNAs coding for the opioid peptide precursors in pituitary and adrenal by "in situ' hybridization: study in several mammal species

The messenger RNAs coding for opioid peptide precursors have been detected and mapped in histological sections by "in situ' hybridization using specific DNA probes labelled with 32P. Using bovine preproenkephalin A (PPA) cDNA, PPA mRNA was detected in adrenal medulla of bull, hamster and guinea pig. No signal was detected in adrenal of man, rat and cat. The pro-opiomelanocortin (POMC) mRNA was detected in pituitary of man, bull, cat, rat and pig, in all cells of the intermediate lobe as well as in scattered cells of the anterior lobe producing POMC. Adequate controls demonstrated the specificity of the labelling. These results provide evidence of the expression of the gene coding for PPA in the adrenal and for POMC in the pituitary. They show cross-hybridization of one DNA probe with mRNAs of various mammals and then provide evidence that one single probe can be used to analyze expression of a given gene in tissues of several animal species by "in situ' hybridization.     

水通道蛋白4在大鼠脑垂体中的表达

目的：研究水通道蛋白4(AQP4)及其mRNA在脑垂体中的表达,探讨其在脑垂体激素分泌过程中的作用。方法：应用免疫组织化学和原位杂交技术,观察成年Wistar大鼠脑垂体中AQP4及其mRNA的正常分布。结果：AQP4及其mRNA在成年大鼠神经垂体的垂体细胞上表达呈阳性,分布在毛细血管窦周围的垂体细胞表达尤为强烈。腺垂体的所有细胞均有AQP4的表达,胞质中AQP4 mRNA表达呈阳性。中间叶所有细胞AQP4及其mRNA的表达呈弱阳性,其中滤泡星形细胞表达较内分泌细胞强烈。结论：AQP4广泛分布于脑垂体的各种组织细胞表面,可能在垂体激素的正常分泌过程中起重要的调节作用。     

Development of thyroid-stimulating hormone beta subunit-producing cells in the chicken embryonic pituitary gland

In previous studies, the distribution of thyrotropes in the chicken pituitary gland has been analyzed by immunohistochemistry using heterologous antibodies. In this study, we examined the distribution of thyroid-stimulating hormone beta subunit-immunopositive (TSHbeta-ip) cells and the expression of TSHbeta mRNA in the pituitary glands of chicken embryos by immunohistochemistry using a specific antiserum to the chicken TSHbeta, in situ hybridization and RT-PCR. Immunohistochemical and morphometric analyses revealed that the TSHbeta-ip cells first appeared on embryonic day 10 (E10) in the pituitary gland and were mainly distributed in the cephalic lobe and that the cell density on E20 was almost 4 times greater than that on E10. The chicken TSHbeta-ip cells could be classified into two types based on morphological characteristics: round-shaped cells and club-shaped cells, which have long cytoplasmic processes. In situ hybridization analysis revealed that TSHbeta mRNA-expressing cells were expressed from E9 in the cephalic lobe and that the extent of TSHbeta mRNA-expressing cells coincided with that of TSHbeta-ip cells. RT-PCR also showed that TSHbeta mRNA was expressed from E9 and that Pit-1 mRNA was expressed from E5. These results clearly demonstrated that the expression of chicken TSHbeta mRNA starts on E9, that TSHbeta-ip cells appear on E10, mainly in the cephalic lobe, and that TSHbeta-ip cells can be classified into two cell types (round- and club-shaped cells).     

The Expression of Wnt4 Is Regulated by Estrogen via an Estrogen Receptor Alpha-dependent Pathway in Rat Pituitary Growth Hormone-producing Cells

Wnt signaling is important in many aspects of cell biology and development. In the mouse female reproductive tract, Wnt4, Wnt5a, and Wnt7a show differential expression during the estrus cycle, suggesting that they participate in female reproductive physiology. Although the pituitary is a major gland regulating reproduction, the molecular mechanism of Wnt signaling here is unclear. We elucidated the subcellular distribution of Wnt4 in the pituitary of estrogen-treated ovariectomized female rats. Expression of Wnt4 mRNA increased dramatically, particularly in proestrus compared with estrus and metestrus. Wnt4 protein was observed in the cytoplasm of almost all growth hormone (GH)-producing cells and in only a few thyroid-stimulating hormone β (TSHβ)-producing cells. In rat GH-producing pituitary tumor (MtT/S) cells, estrogen-induced expression of Wnt4 mRNA was completely inhibited by estrogen receptor antagonist ICI 182,780 in vitro. Thus, rat pituitary GH cells synthesize Wnt4 and this is induced by estrogen mediated via an estrogen receptor alpha-dependent pathway.     

分泌颗粒素Ⅱ和促肾上腺皮质激素在脑损伤大鼠垂体腺细胞共存(英文)

采用荧光双标记技术研究了脑损伤时大鼠垂体前叶促肾上腺皮质激素和分泌颗粒素 的定位。垂体前叶细胞呈现分泌颗粒素 染色强阳性 ,阳性物质位于胞浆中。免疫荧光双标记技术用于鉴定分泌颗粒素 和促肾上腺皮质激素共存的细胞 ,共聚焦显微镜观察显示促肾上腺皮质激素和分泌颗粒素 共存于同一细胞。结果表明 :分泌颗粒素 可在大鼠垂体前叶促肾上腺皮质激素阳性细胞表达 ,其生理作用可能是调节分泌泡的 p H值以利于促肾上腺皮质激素从其前体裂解。     

Effect of aging on the expression of BDNF and TrkB isoforms in rat pituitary

Brain-derived neurotrophic factor (BDNF) is a key regulator of neuronal plasticity in adult rat brain and its effects are mediated through TrkB receptors. BDNF and its receptors are also localized in the pituitary, but their expressions throughout the rat lifespan are poorly known. Here we analyzed levels of BDNF and the different subtypes of TrkB receptors (mRNA and proteins) in the rat pituitary at different stages of life. BDNF immunoreactivity was expressed in folliculo-stellate cells from the anterior pituitary and in the intermediate lobe. TrkB.FL and TrkB.T1 receptors were strongly and essentially expressed in the intermediate lobe similar to the alpha-MSH localization pattern. These receptors begun decreasing at middle-age but TrkB.T2 was not detected in the pituitary at any age. Finally, in vitro alpha-MSH release from the intermediate lobe was correlated with the receptor content throughout the lifespan. The present results demonstrate the presence of BDNF in folliculo-stellate cells and indicated that receptors, rather than BDNF itself, are impaired with aging. These changes can contribute to explain age-dependent endocrine changes.     

Postnatal changes in expression of vesicular glutamate transporters in the main olfactory bulb of the rat

Olfactory information is initially processed through intricate synaptic interactions between glutamatergic projection neurons and GABAergic interneurons in the olfactory bulb. Although bulbar neurons and networks have been reported to develop even postnatally, much is yet unknown about the glutamatergic neuron development. To address this issue, we studied the postnatal ontogeny of vesicular glutamate transporters (VGLUT1 and VGLUT2) in the main olfactory bulb of rats, using in situ hybridization, immunohistochemistry, and their combination. In situ hybridization data showed that VGLUT1 mRNA is intensely expressed in differentiating mitral cells and smaller cells of the mitral cell layer (MCL) on postnatal day 1 (P1), and also at lower levels in small- and medium-sized cells, presumably tufted cell populations, of the external plexiform layer (EPL) from P5 onward. VGLUT2 mRNA was expressed in many MCL cell populations on P1, also in small- and medium-sized cells of the EPL at almost the same level as MCL cells between P5 and P7, and became apparently less intense in the MCL than in the EPL from P10 onward. The expression, unlike VGLUT1 mRNA, was also found in small-sized cells of the interglomerular region. In partial agreement with these data, immunohistochemical analyses demonstrated that subsets of mitral and EPL cells are stained for VGLUT1 or VGLUT2, with the former cells coexpressing both subtypes until P5. Moreover, a combined fluorescence in situ hybridization–immunohistochemical dual labeling of the P10 bulb revealed that neither VGLUT1 nor VGLUT2 mRNA is expressed in GABAergic or dopaminergic periglomerular cells, implying their expression in other periglomerular cell subclasses, external tufted cells and/or short-axon cells. Thus, the present study suggests that early in the postnatal development distinct glutamatergic bulbar neurons of rats express spatiotemporally either or both of the two VGLUT subtypes as a specific vesicular transport system, specifically contributing to glutamate-mediated neurobiological events.     

In situ hybridization and immunohistochemistry for the detection of porcine cytomegalovirus

To establish in situ hybridization and immunohistochemistry based-assays for the detection of porcine cytomegalovirus, routinely processed renal tissue sections from 34 diseased piglets suspected of having the infection were obtained and examined. Using hematoxylin and eosin, porcine cytomegalovirus inclusion bodies were found in the nucleus of renal epithelial cells and capillary endothelial cells in the renal medulla in 30 cases. Inclusion bodies corresponding to porcine cytomegalovirus mRNA after in situ hybridization or porcine cytomegalovirus antigens after immunohistochemistry were easily determined. The cells were characterized by cytomegaly and basophilic intranuclear inclusion bodies. Using in situ hybridization, porcine cytomegalovirus mRNA were clearly detected in the nucleus and cytoplasm of the cells in 28 of the 30 (93.3%) cases. Using immunohistochemistry, porcine cytomegalovirus antigens were clearly detected in the cytoplasm of the cells in 21 of the 30 (70.0%) cases. Higher specificities and increased intensity of staining was observed with minimal background using in situ hybridization and immunohistochemistry compared with hematoxylin and eosin. Thus, the two established methods are useful and helpful tools for detecting the presence of a porcine cytomegalovirus infection.     

GnRH receptor mRNA expression by in-situ hybridization in the primate pituitary and ovary

Gonadotrophin-releasing hormone (GnRH) receptors are presenton the ovary as well as in the anterior pituitary gland. GnRHanalogues may exert their actions in part via these ovarianreceptors. However, in the primate ovary, GnRH receptors areof low affinity and their significance is questionable. Theaim of the present study was to compare pituitary and ovarianexpression of the GnRH receptor mRNA by in-situ hybridizationto gain further information on the possible significance ofthe ovarian receptor. Pituitaries and ovaries were obtainedfrom two stump-tailed macaque monkeys and three marmoset monkeysat the mid-luteal phase of the ovulatory cycle. Human corporalutea were obtained during the early and mid-luteal phase andafter ‘rescue’ by human chorionic gonadotrophin(HCG) and a whole ovary obtained during the late luteal phase(n=1 per group). Frozen tissue sections were incubated witha 33P-labelled probe to the human GnRH receptor and exposedfor 4 weeks. All pituitary glands exhibited intense silver grainsin the anterior pituitary gland. In the ovaries, grains werepresent at low levels in the granulosa cells of antral follicles,just above tissue background in corpora lutea and indistinguishablefrom tissue background in the remaining ovarian compartments.These results demonstrate that the GnRH receptor mRNA in theprimate pituitary is present in sufficient quantities to beclearly detectable in the anterior pituitary gland by in-situhybridization. In contrast, in the human and monkey, ovary levelsof mRNA appear to be very low. corpus luteum/follicle/GnRH mRNA/in-situ hybridization     

Growth hormone-releasing hormone receptor mRNA in acromegalic pituitary tumors.

The growth hormone (GH)-releasing hormone receptor (GHRH-R) has been recently cloned and found to be a member of a new family of seven transmembrane receptors that includes secretin, vasoactive intestinal peptide, calcitonin, and corticotropin-releasing factor. GHRH-R mRNA has been demonstrated by Northern blot analyses to be present specifically in the anterior pituitary gland. To determine the precise cellular localization of this receptor in normal anterior pituitary and pituitary adenomas, GHRH-R mRNA was analyzed in 2 normal human pituitary glands and 16 human pituitary adenomas using in situ hybridization. GHRH-R was specifically localized in somatotroph cells in the normal pituitary. In the adenomas, all GH-producing adenomas originating from acromegalic patients demonstrated up-regulation of GHRH-R mRNA when compared with levels in the normal pituitary. Only one of five clinically nonfunctioning adenomas, a gonadotroph luteinizing hormone/follicle-stimulating hormone-positive adenoma, exhibited up-regulation of this receptor message. Adrenocorticotrophic hormone-secreting and prolactin-secreting adenomas did not express GHRH-R message. In summary, GHRH-R is specifically expressed in somatotrophs and GH-producing adenomas, suggesting that GHRH-R may influence GH release in adenomas similar to this receptor's actions in the normal somatotrophs and may be involved in the growth of GH-secreting adenomas.     

Electron Microscopic and Confocal Laser Scanning Microscopic Observation of Subcellular Organelles and Pituitary Hormone mRNA: Application of Ultrastructural In Situ Hybridization and Immunohistochemistry to the Pathophysiological Studies of Pituitary Cells

Nonradioisotopic electron microscopic (EM)in situ hybridization (ISH) (EM-ISH) with biotinylated oligonucleotide probes is utilized for the ultrastructural visualization of pituitary hormone mRNA in rat pituitary cells. EM-ISH is an important tool for clarifying the intracellular localization of mRNA and the exact site of specific hormone synthesis on the rough endoplasmic reticulum. The simultaneous visualization of mRNA and encoded protein in the same cell using preembedding EM-ISH and subsequent postembedding immunoreaction with protein A colloidal gold complex can provide an important clue for elucidating the intracellular correlation of mRNA translation and secretion of translated protein. Another focus of this review is the utilization of a recently developed imaging system of confocal laser scanning microscopy (CLSM). The combination of CLSM and image analysis system (IAS) enables us to visualize an individual dimensional image of the intracellular distribution of mRNA and subcellular organelles successfully at any optional cross sections of light microscopic ISH studies, and can be another useful tool for the ultrastructural ISH study of mRNA.     

Histologic Distribution,Fragment Cloning,and Sequence Analysis of G Protein Couple Receptor 30 in Rat Submaxillary Gland

Recent studies indicated that G protein couple receptor 30 (GPR30), a nongenomic estrogen receptor, is widely expressed in many organ systems inducing many quick reaction of estrogen. However, there was rare report about the expression of GPR30 in the salivary gland. In the present study, we investigated the distribution of GPR30 in rat submaxillary gland by means of immunohistochemistry and in situ hybridization. GPR30 core sequences were amplified by RT‐PCR with total RNA extracted from rat submaxillary gland and were analyzed by sequencing with Sanger's method. The results showed that the epithelial cells of serous alveoli and granular convoluted duct in rat submaxillary gland displayed GPR30‐immunoreactivity on the plasma membrane and cytoplasm. Moreover, GPR30 mRNA hybridization signals were also detected in the cytoplasm of the above cells. GPR30 cDNA sequence cloned from rat submaxillary gland is identical to that of GPR30 from rat paraventricular and supraoptic nucleus. In conclusion, the expression of GPR30 in the serous and granular epithelial cells of submaxillary gland indicates that submaxillary gland could also be a target organ rapidly responding to estrogen stimulus, and estrogen may be involved in the functional regulation of submaxillary gland. Anat Rec, 2011. © 2011 Wiley‐Liss, Inc.