Keiichi Kawamoto

Type IV collagenase activity and cavernous sinus invasion in human pituitary adenomas

SummaryPituitary adenomas are regarded generally as benign tumours, but some of them can invade the cavernous sinus. On the other hand, matrix metalloproteinase-9 (MMP-9), which is a type IV collagenase, recently has been found to be expressed in matastases and to be related to the invasiveness of various malignant tumours including brain tumours. In order to investigate some characteristic features of pituitary adenomas which invade the cavernous sinus, we examined immunohistological studies for MMP-9 in seven pituitary adenomas for type IV collagen in a dura mater and assayed for type IV collagenase activity in seven adenomas using type IV collagen labelled with fluorescein isothiocyaniate (FITC).We found immunopositive adenoma cells for MMP-9 in all invasive adenoma and immunopositive spindle like cells for type IV collagen in the dura mater. All three invasive adenomas had high levels of type IV collagenase activity (0.57–0.72 U/ml), but the four adenomas which did not invade the cavernous sinus had low levels of type IV collagenase activity (0.0–0.10 U/ml).These results suggest that the level of type IV collagenase activity in a pituitary adenoma may be related to its ability to invade the cavernous sinus.

Analysis of the growth rate and cavernous sinus invasion of pituitary adenomas

SummaryPituitary adenomas generally are regarded as benign tumours, but a part of them can invade the cavernous sinus and recur. We examined 43 pituitary adenomas for the following factors: tumour volume, endocrinological function, cavernous sinus invasion, and growth rates examined by using anti-proliferating cell nuclear antigen (PCNA) and MIB1 (a novel anti-Ki-67) as markers.There was significant correlation between PCNA- and MIB1-positive cell rates and PCNA- and MIB1-positive cell rates were higher in the three cases with rapid regrowth than in the other cases. Staining was stronger and more distinct for MIB1 than for anti-PCNA; thus, MIB1-positive cells were easily distinguished by their intense immunoreactivity. MIB1 may be useful for detecting those rare cases with rapid regrowth even when initially regarded as benign tumours.Adenomas with cavernous sinus invasion were significantly larger than those demonstrating no invasion. However, no significant difference was found in the frequency of PCNA- or MIB1-positive cells between adenomas with and without cavernous sinus invasion.These findings suggest that cavernous sinus invasion and growth rate are independent biological factors. Therefore, cavernous sinus invasion may be due to chemical factors produced by the tumour itself rather than as a result of rapid tumour growth.

Matrix metalloproteinase-9 secretion by human pituitary adenomas detected by cell immunoblot analysis

SummaryTwenty-two pituitary adenomas were examined on the secretion of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) using a cell immunoblot assay, and discussed regarding an association between cavernous sinus invasion and the secretion of these proteins. The cell immunoblot assay, a kind of immunoblot procedure, is able to detect proteins at the single cell level and to detect the incidence of tumour cells secreting the target proteins in the total tumour cell population. The incidence of tumour cells secreting MMP-9 was significantly higher in invasive adenomas than in noninvasive ones. On the other hand, TIMP-1 secretion was not detected in any adenomas in this study. This result suggested that MMP-9 secretion, and especially the number of MMP-9-secreting cells, may be associated with cavernous sinus invasion of pituitary adenomas.

Effect of pituitary adenylate cyclase-activating polypeptide (PACAP) on prolactin and somatolactin release from the goldfish pituitary in vitro

Pituitary adenylate cyclase-activating polypeptide (PACAP) plays a role in mediating growth hormone and gonadotropin release in the teleost pituitary. In the present study, we examined the immunohistochemical relationship between PACAP nerve fibers and prolactin (PRL)- and somatolactin (SL)-producing cells in the goldfish pituitary. Nerve fibers with PACAP-like immunoreactivity (PACAP-LI) were identified in the neurohypophysis in close proximity to cells containing PRL-LI or SL-LI. Several cells with PRL-LI or SL-LI showed PACAP receptor (PAC(1)R)-LI. The cell immunoblot assay method was used to examine the effect of PACAP on PRL and SL release from dispersed goldfish pituitary cells. Treatment with PACAP increased the immunoblot area for PRL- and SL-LI from individual pituitary cells in a dose-dependent manner. The effect of PACAP on the expression of mRNAs for PRL and SL in cultured pituitary cells was also tested. Semiquantitative analysis revealed that the expression of SL mRNA, but not PRL mRNA, was increased significantly by the treatment with PACAP. The effect of PACAP on intracellular calcium mobilization in isolated pituitary cells was also investigated using confocal laser-scanning microscopy. The amplitude of Ca(2+) mobilization in individual cells showing PRL- or SL-LI was increased significantly following exposure of cells to PACAP. These results indicate that PACAP can potentially function as a hypophysiotropic factor mediating PRL and SL release in the goldfish pituitary.

Vasoactive intestinal peptide- and peptide histidine isoleucine amide-like immunoreactivity colocalize with vasopressin-like immunoreactivity in the canine hypothalamo-neurohypophysial neuronal system.

The distribution of vasoactive intestinal peptide (VIP) and peptide histidine isoleucine amide (PHI) was investigated in the canine hypothalamus by immunocytochemistry. VIP- and PHI-like immunoreactive neurons were detected in the magnocellular supraoptic and paraventricular nucleus. These magnocellular VIP- and PHI-producing neurons coexist with vasopressin-like immunoreactivity and send axons to the median eminence and neurohypophysis. These findings may serve as an anatomical basis for studying the function of VIP and PHI on pituitary hormone secretion.

Endocrine Control of the Reproductive Activity in Hibernating Bats

Abstract Bats, Chiroptera, constitute the second largest order of the class Mammalia and vary greatly in habitats, available foods and mating systems. The timing, duration and patterns of reproduction in bats vary considerably among species and different localities. Though much is known about the reproductive phenomena and associated endocrine characteristics of various species, the central mechanism regulating the peculiar delay and asynchrony in reproductive activity remains to be elucidated. The current understanding on the endocrine characteristics and possible mechanism of regulation of the hypothalamo-adenohypophysial-gonadal axis of bats will be reviewed, based mainly on our own studies in hibernating rhinolophid bats.

Plasticity of vasopressin- and oxytocin-containing fibers in the median eminence in hypophysectomized young and old mice

Rearrangements of vasopressin- and oxytocin-containing fibers in the external layer of the median eminence after hypophysectomy were compared between young and old mice. In 3-month-old hypophysectomized mice, the increase in the number of fibers containing vasopressin was greater than that observed in 19-month-old hypophysectomized ones, suggesting a decrease in axonal plasticity in old mice. No difference with age was detected for the plasticity of fibers containing oxytocin.

Ultrastructural Changes and Proliferation of Pituicytes in Mouse Posterior Lobe during Water Deprivation and Rehydration

Ultrastructural changes and proliferation of pituicytes during water deprivation and rehydration were studied in the posterior lobe of C57BL/Tw mice. Deprivation for 3 days brought about a significant increase in the number of electron-dense bodies (lysosomes) in pituicyte perikarya and their processes. The frequency of pituicytes enclosing neurosecretory axons in their cytoplasm significantly decreased as compared with that of the controls. 12-hour rehydration following deprivation for 3 days induced extensive development of rough endoplasmic reticulum and Golgi apparatus and an increase in frequency of neurosecretory axons enwrapped by pituicyte cytoplasm. However, at 2 days of rehydration the morphology of pituicytes was no more different from that of the controls. Mitotic figures of pituicytes were not encountered throughout the deprivation period of 6 days, but rehydration for 12 h and 1, 2, or 3 days following deprivation for 3 or 6 days was effective in eliciting an increase in mitotic activity. The present results indicate that pituicytes in the mouse posterior lobe are intimately related with the secretory mechanism of neurosecretory material from the neurosecretory axons and that the proliferation of pituicytes is stimulated in conditions of reaccumulation of neurosecretory material.

Regeneration of neurohypophyseal hormone-producing neurons in hypophysectomized immature rats

Age-related changes in the regeneration of vasopressin (AVP) and oxytocin (OXT) axons after hypophysectomy in rats was immunohistochemically examined. Rats were hypophysectomized at 9, 16, 23, 30 and 90 days of age, and sacrificed 10 days after the operation. AVP or OXT immunoreactivity in the external layer of the median eminence (ME) was generally stronger in hypophysectomized immature rats than in hypophysectomized adult rats, and the age-related difference in immunoreactivity was more conspicuous for AVP axons than OXT ones. The cell body size of AVP or OXT neurons in hypophysectomized adult rats was not significantly different from the value of unoperated or initial control rats. However, the neurons in immature rats became significantly larger after hypophysectomy, compared with those of initial controls. These results indicate that AVP- and OXT-producing neurons in immature rats, as early as at 9 days of age, are endowed with the capacity of axonal rearrangement to the external layer of the ME after hypophysectomy, and that the stronger immunoreactivity in the external layer of the ME in immature rats than in the adult may be due to the differences in the rate of synthesis of neurohypophyseal hormones and the regenerative potency of neurons.

Secretory activity of gonadotropin and the responsiveness of gonadotrophs to gonadotropin-releasing hormone during the annual reproductive cycle of male bats, Rhinolophus ferrumequinum: Analysis by cell immunoblot assay

The purpose of this study was to examine secretory activity of gonadotropin (Gn) and the responsiveness of Gn secretion to Gn-releasing hormone (GnRH) in male horseshoe bats, Rhinolophus ferrumequinum, during the annual reproductive cycle. Anterior pituitary cells were monodispersed and subjected to cell immunoblot assay for Gn. Cell blots specific for follicle stimulating hormone (FSH) or luteinizing hormone (LH) were quantified using a microscopic image analyzer. The percentages of LH- or FSH-secreting cells detected as immunoreactive cell blots were markedly increased in the spermatogenic period (summer) and decreased in the hibernation period (winter). The mean Gn secretion from individual cells and total Gn secretion per unit area of the transfer membrane also showed similar changes. The responsiveness of Gn secretion to GnRH was greater in the spermatogenic period than in other seasons. On the other hand, although the secretory activity of Gn was markedly decreased during hibernation, a stimulatory effect of GnRH on Gn secretion was observed. These findings suggest that seasonal changes in the release of Gn required for gametogenesis and gonadal steroidogenesis varied depending on the reproductive activity and seasonal changes in Gn sensitivity to stimulatory effects of GnRH due to alterations in GnRH receptor numbers and/or in postreceptor events of gonadotrophs.