To clarify the acute and chronic effects of GnRH agonists on pituitary gonadotropes, changes in the ultrastructure of male rat gonadotropes were examined immunocytochemically and morphometrically after the administration of a one-month depot formulation of the GnRH agonist, leuprorelin. Immediately after the depot administration, the relative amounts of secretory granules drastically decreased in gonadotropes concomitantly with a marked increase in the plasma LH level. After the acute hyperstimulated phase, secretory granules in gonadotropes were gradually restored although the newly synthesized granules were less densely immunolabeled for LHβ; their relative amounts and sizes were still significantly smaller than the controls after depot treatment for 28 days. Eighty-four days after the leuprorelin depot administration, however, the ultrastructural characteristics of pituitary gonadotropes appeared to recover as observed in controls: there were no significant differences in the relative amounts, sizes, and labeling densities for LHβ of secretory granules, and the amounts of chromogranin A (CgA) and secretogranin II (SgII) were restored in secretory granules to control levels. When the rats were repeatedly treated with the leuprorelin depot at intervals of 4 weeks, the expression and intracellular storage levels of gonadotropins remained highly suppressed, judging from the labeling density for LHβ. These findings suggest that the depot formulation of the GnRH agonist could suppress both the biosynthesis and release of gonadotropins for a month by synergistically depleting the intracellular storage of secretory granules at the onset of the treatment and by inducing the subsequent desensitization of the GnRH receptor signaling.

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The expression of the cytoglobin/stellate cell activation-associated protein (Cygb/STAP) was recently confirmed in all splanchnic vitamin A-storing cells - including hepatic stellate cells (HSCs) - in normal conditions. In the hepatic fibrous lesion, the expression of Cygb/STAP has been shown to be upregulated in activated HSCs and myofibroblasts (MFs), which have synthesized extracellular matrices. Furthermore, splanchnic vitamin A-storing cells have been reported to be distributed in the kidney. In this study, we clarify the contribution of vitamin A-storing cells to renal fibrosis by focusing on Cygb/ STAP. Adult mice were subjected to unilateral ureteral obstruction (UUO) and kidneys were harvested 1, 3, 7, and 10 days after UUO.
Numbers of Cygb/STAP-immunopositive cells as well as Cygb/STAP mRNA 3 days after UUO (UUO day 3 kidney) increased. Vitamin A-autofluorescence was observed in intertubular spaces of controls but gradually declined in a time-dependent manner after UUO. Cygb/STAP⁺ cells were not completely identical with α-smooth muscle actin (αSMA)-positive cells in the control or UUO day 7 kidneys. Immunohistochemical analysis for Cygb/STAP and fibulin-2 (Fib), a specific marker for distinguishing MFs from activated HSCs, revealed that the number of Fib⁺STAP⁺ cells (MFs) and Fib-STAP⁺ cells (splanchnic vitamin A-storing cells) significantly increased in UUO day 3 and UUO day 7 kidneys compared with the controls. Our present findings support the concept that Cygb/STAP can be a unique marker for splanchnic fibroblast-like cells, namely the vitamin A-storing cell lineage, and suggest that splanchnic vitamin A-storing cells contribute to renal fibrogenesis in the obstructed kidney.

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The ultrastructure of the rat intestinal interstitium with regard to the mechanical components was analyzed from a functional viewpoint utilizing serial horizontal as well as longitudinal sections through the lamina propria mucosae, including both villi and crypts. The axial smooth muscle cells in the villi (villus-axial SMs) exhibited different configurations at various levels of the wall. They were separated from the voluminous fluid-filled spaces by sheet-like processes of fibroblasts in the upper part of the intravillous interstitium, formed a sheet around the central lymphatics, and were covered by the sheet-like processes of fibroblasts in the lower part of the intravillous interstitium. These villus-axial SMs were poorly developed and associated with the lymphatic walls in the upper part of the pericryptal interstitium; they were tapered and connected to microtendons composed of fascicles of longitudinal collagen fibrils in the lower part of pericryptal interstitium. At the apical termination, the villus-axial SMs were connected to myofibloblasts, which sent off many processes into the subepithelial meshwork layer of fine cell processes and extracellular matrices. The villus-axial SMs possibly develop longitudinal tension against the intravillous hydraulic pressure developing from the transepithelial absorption through the intestinal epithelium.

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Neurotrophic factors are thought to function in the survival and maintenance of the taste buds and nerve fibers innervating them. Laser capture microdissection (LCM) coupled with the reverse transcription polymerase chain reaction (RT-PCR) was performed to detect the mRNA of neurotrophic factors and their receptors in the taste buds of adult mouse circumvallate papillae. Results showed mRNAs of the ciliary neurotrophic factor (CNTF), its receptor (CNTFR), glial cell line-derived neurotrophic factor (GDNF), GDNF family receptors α-1 (GFRα-1), GFRα-2, and RET tyrosine kinase receptor (RET), neurotrophin (NT)3, NT4/5, tyrosine kinase (Trk) C, nerve growth factor (NGF), and TrkA were expressed in the isolated taste buds. Among these neurotrophic factors, GDNF, GFRα-1, GFRα-2, NT3, NT4/5, NGF, and TrkA were previously found in the taste buds immunohistochemically and were detected at the mRNA level in the present study. The present immunohistochemical study revealed that CNTF, CNTFR, and the RET tyrosine kinase receptor, which binds GDNF family/ receptor complexes, were also expressed in the taste buds. However, by in situ hybridization, mRNAs of CNTF and RET were not detected in the taste buds from adult mice although they were found in those from early postnatal mice. CNTFR mRNA did not show any specific pattern in the taste buds. Moreover, mRNA expressions of NT4/5 and TrkC was re-examined by in situ hybridization; however no specific pattern was found for them in the taste buds. In summary, LCM is a useful tool for the detection of a relatively small amount of mRNA, such as that of neurotrophic factors and receptors in the taste buds.

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D-Serine is an endogenous and obligatory coagonist for the glycine site of the N-methyl-D-aspartate receptor in the mammalian brain. D-Serine is synthesized from L-serine by serine racemase; immunohistochemical studies have long been believed to indicate that serine racemase and D-serine occur predominantly in astrocytes. However, we have recently demonstrated in the primary cultures that both the mRNA and protein levels of serine racemase are higher in neurons than in astrocytes. Here we report the application of in situ hybridization based on tyramide signal amplification for the detection of serine racemase mRNA in sections of the adult rat brain. Serine racemase mRNA could be demonstrated in a large number of neurons throughout the brain, especially in the forebrain such as the cerebral cortex, striatum, and hippocampus. This is the first study to demonstrate the exact localization of serine racemase mRNA at the cellular or tissue level. These results suggest that neuron-derived D-serine could modulate neurotransmission via the glycine site of N-methyl-D-aspartate receptors.

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The intestinal mucosa is vulnerable to an ischemia-reperfusion (I/R) attendant on some bowel diseases and surgery; thus, the restoration of the mucosal integrity is critical to achieving functional recovery of the intestine injured by I/R. In this histochemical study, we investigated the alteration of the central lacteals - which are essential for the transport of fat, tissue fluid, and immune cells in the intestinal mucosa - in the murine jejunum after I/R. The intestine inflicted with I/R demonstrated mucosal injury involving the inflammatory response, with interstitial edema, disruption of the villous tissue, and subsequent tissue regeneration of the villi. The regenerative villous tissue revealed lymphatic regrowth showing proliferative activity from the residual mucosal lymphatics behind the regenerated blood vasculature. During the regenerative phase, the blood vascular pericytes expressed an intense immunoreaction for VEGF-A, an inducer for monocyte/macrophage recruitment as well as angiogenesis. Also, the F4/80-immunopositive macrophages significantly increased in number in the regenerating villous stroma. Furthermore, the macrophages recruited around the regrowing lacteals expressed the immunoreactivity for VEGF-C, which is a highly specific lymphangiogenic factor. The present study is first to delineate alterations in the central lacteals in the small intestine following I/R, thereby suggesting that the recruitment of the macrophages induced by upregulation of VEGF-A in the pericytes of regenerative blood vessels might promote reconstruction of the central lacteals through their release of VEGF-C.

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