Manabu Matsuda

Serotonin regulates mammary gland development via an autocrine-paracrine loop.

Mammary gland development is controlled by a dynamic interplay between endocrine hormones and locally produced factors. Biogenic monoamines (serotonin, dopamine, norepinephrine, and others) are an important class of bioregulatory molecules that have not been shown to participate in mammary development. Here we show that mammary glands stimulated by prolactin (PRL) express genes essential for serotonin biosynthesis (tryptophan hydroxylase [TPH] and aromatic amine decarboxylase). TPH mRNA was elevated during pregnancy and lactation, and serotonin was detected in the mammary epithelium and in milk. TPH was induced by PRL in mammosphere cultures and by milk stasis in nursing dams, suggesting that the gene is controlled by milk filling in the alveoli. Serotonin suppressed beta-casein gene expression and caused shrinkage of mammary alveoli. Conversely, TPH1 gene disruption or antiserotonergic drugs resulted in enhanced secretory features and alveolar dilation. Thus, autocrine-paracrine serotonin signaling is an important regulator of mammary homeostasis and early involution.

Expression and function of cystine/glutamate transporter in neutrophils

Reactive oxygen species (ROS) produced by neutrophils are essential in the host defense against infections but may be harmful to neutrophils themselves. Glutathione (GSH) plays a pivotal role in protecting cells against ROS‐mediated oxidant injury. Cystine/glutamate transporter, designated as system xc– and consisting of two proteins, xCT and 4F2hc, is important to maintain GSH levels in mammalian‐cultured cells. In the present paper, we have investigated system xc– in neutrophils. In human peripheral blood neutrophils, neither the activity of system xc– nor xCT mRNA was detected. The activity was induced, and xCT mRNA was expressed when they were cultured in vitro. The mRNA expression was much enhanced in the presence of opsonized zymosan or PMA. In contrast, mouse peritoneal exudate neutrophils, immediately after preparation, exhibited system xc– activity and expressed xCT mRNA. The activity and the expression were heightened further when they were cultured. Peritoneal exudate cells (mostly neutrophils) from xCT‐deficient (xCT−/−) mice had lower cysteine content than those from the wild‐type mice. GSH levels in the xCT−/−cells decreased rapidly when they were cultured, whereas those in the wild‐type cells were maintained during the culture. Apoptosis induced in culture was enhanced in the xCT−/−cells compared with the wild‐type cells. These results suggest that system xc– plays an important role in neutrophils when they are activated, and their GSH consumption is accelerated.

Changes in Expression of Prolactin- and Cortisol-receptor Genes during Early-life Stages of Euryhaline Tilapia (Oreochromis mossambicus) in Fresh Water and Seawater

Abstract Expression of prolactin receptor (PRLR) and cortisol receptor (CR) mRNAs was examined during early-life stages of euryhaline Mozambique tilapia (Oreochromis mossambicus) by competitive reverse transcription-polymerase chain reaction (cRT-PCR). Concentration of prolactin receptor mRNA was higher in the gills of mature fish reared in fresh water (FW) than in those reared in seawater (SW), whereas no difference was seen in CR mRNA. Whole eggs just after fertilization contained the receptor mRNAs for both prolactin and cortisol. The concentration of PRLR mRNA increased gradually as the embryo grows both in FW and in SW. On the other hand, the concentration of CR mRNA was highest in the egg just after fertilization, decreased rapidly toward hatching, and increased slightly thereafter. When embryos 3 days before hatching were transferred to SW, the levels of PRLR mRNA were significantly lower at the time of hatching and also 3 days after hatching than in the embryo and larvae maintained in FW. Environmental salinity did not affect CR mRNA content at any stage examined. Both PRLR and CR mRNAs were identified in the yolk-sac membrane and in the embryonic body. Significantly more PRLR gene was expressed in the embryonic body developing in FW than in SW, whereas no difference was seen in the yolk-sac membrane. The greater expression of PRLR gene in embryos and larvae developing in FW than in those in SW clearly indicates the presence of regulatory mechanisms of gene expression in early-life stages of tilapia.

Suppression of the development of experimentally induced uterine adenomyosis by a novel matrix metalloproteinase inhibitor, ONO-4817, in mice.

The inhibitory effects of a novel, orally active matrix metalloproteinase (MMP) inhibitor, ONO-4817, on the development of uterine adenomyosis induced experimentally by pituitary grafting were examined in mice. Mice were given transplants of isologous anterior pituitary glands (PGs) into the right uterine lumen at 7 weeks of age and were fed chow containing 0.1% to 1.0% ONO-4817 from 8 to 14 weeks of age. Mice treated with 0.3% or 1.0% ONO-4817 showed a significantly lower incidence of the development of adenomyosis than vehicle-treated mice. To evaluate the inhibitory effects of ONO-4817 on the progression of the invasion of the adenomyotic tissues, mice receiving PG grafts at 7 weeks of age were treated with 1.0% ONO-4817 from 13 to 17 weeks of age. The degree of pathological progression of adenomyosis was graded from 1 to 5 in increments of 1. The degree of the progression of the lesion was less in the uteri exposed to ONO-4817 (2.71 ± 0.93) than in the uteri not exposed to the inhibitor (4.33 ± 0.75). Finally, the invasiveness of endometrial stromal cells obtained from adenomyotic uteri into Matrigel consisting mainly of type IV collagen and laminin was examined using an invasion assay. The assay showed that the treatment with ONO-4817 markedly suppressed the invasion of the stromal cells of the adenomyotic uteri into the gel. These results indicate that ONO-4817 may be an effective inhibitor of the development of adenomyosis.

Extrapituitary Expression of the Prolactin Gene in the Goldfish, African Clawed Frog and Mouse

Abstract In mammals, the pituitary hormone prolactin is also produced in various extrapituitary tissues and may act in an auto/paracrine fashion. To explore the comparative aspects of extrapituitary prolactin, the distributions of prolactin transcripts were investigated in extrapituitary organs of the goldfish, African clawed frog and mouse by reverse transcription-polymerase chain reaction (RT-PCR). For comparison, the amount of the transcript in mouse tissues was also estimated using competitive RT-PCR. In the goldfish, the transcript was detected in the ovary, testis, liver, kidney, spleen, gill, muscle and brain in slightly lower abundance than in the pituitary, but not detected in the intestine. In the frog, the transcript was detected in the following organs with an order of abundance: pituitary >> brain > testis and ovary. In the mouse, the transcript was detected in the pituitary, brain, testis, and ovary, and its copy number per μg of total RNA was estimated at ~1010 in the male pituitary, ~104 in the placenta, hypothalamus and testis, ~103 in the thymus and experimentally induced deciduoma, and ~102 in the ovary. These results suggest that the origin of extrapituitary prolactin goes back to the common ancestor of fish and tetrapods, but that distinct evolution has occurred in each lineage. The significance of extrapituitary PRL in non-mammalian species is also suggested.

Chronic effect of hyperprolactinemia on blood glucose and lipid levels in mice.

We studied the chronic effects of hyperprolactinemia, induced by ectopic pituitary grafting, on blood glucose and lipid levels in adult male mice. For one year after pituitary grafting, we measured the blood levels of prolactin, growth hormone (GH), insulin, glucose and free fatty acid (FFA) at various intervals. The graft caused consistent hyperprolactinemia without changes in the serum GH levels. Hypoglycemia developed at 1 and 3 months after grafting but was not accompanied by any changes of the serum insulin levels. Thereafter, the blood glucose and serum insulin levels began to increase in the pituitary-grafted (PG) mice, and at 12 months after the operation, both levels became significantly higher in PG mice than controls. The serum FFA levels and the weight of epididymal fat bodies were significantly lower in PG mice than controls from 3-12 months after the grafting. Thus, hyperprolactinemia leads to persistent hypolipidemia and biphasic changes in the blood glucose level.

Effect of Estrogen on Hyperprolactinemia-Induced Glucose Intolerance in SHN Mice

Abstract The effects of prolactin (PRL) on circulating levels of glucose and insulin, and of estradiol on hyperprolactinemia-induced glucose intolerance of tissues were studied in pituitary-grafted SHN mice (PG mice) and sham-operated controls. Pituitary grafting (PG) decreased blood glucose levels in male mice at 1 and 3 months after the operation but did not alter those in females. PG had little effect on serum insulin levels in males, but increased those in females. In female mice at 2 months after PG, blood glucose levels were significantly higher at 1, 2, and 4 hr after glucose load when compared with those in controls. In contrast, there was no significant difference in blood glucose levels after glucose load between male PG and control mice. The rate at which blood glucose levels decreased was slower in female PG mice than in controls during the 30 min after insulin injection, whereas there was no difference in the rate after insulin injection between male PG and control mice. In ovariectomized (Ovx) mice, no significant difference was found in the blood glucose levels after a glucose load between PG and control groups at 2 months after PG. In Ovx mice treated daily with estrogen, however, a PG-dependent high level of blood glucose was observed after glucose load. These results suggest that hyperprolactinemia decreases glucose tolerance via an increase in insulin resistance in female SHN mice and that estrogen is essential to the expression of the PRL effect.

Water Adaptation Strategy in Anuran Amphibians: Molecular Diversity of Aquaporin

Most adult anuran amphibians except for the aquatic species absorb water across the ventral pelvic skin and reabsorb it from urine in the urinary bladder. Many terrestrial and arboreal species use a region in the posterior or pelvic region of the ventral skin that is specialized for rapid rehydration from shallow water sources or moist substrates. Periods of terrestrial activity can be prolonged by reabsorption of dilute urine from the urinary bladder. Aquaporin (AQP), a water channel protein, plays a fundamental role in these water absorption/reabsorption processes, which are regulated by antidiuretic hormone. Characterization of AQPs from various anurans revealed that the unique water homeostasis is basically mediated by two types of anuran-specific AQPs, i.e. ventral pelvic skin and urinary bladder type, respectively. The bladder-type AQP is further expressed in the pelvic skin of terrestrial and arboreal species, together with the pelvic skin-type AQP. In contrast, the pelvic skin-type AQP (AQP-x3) of the aquatic Xenopus has lost the ability of efficient protein production. The extra C-terminal tail in AQP-x3 consisting of 33 nucleotides within the coding region appears to participate in the posttranscriptional regulation of AQP-x3 gene expression by attenuating protein expression. The positive transcriptional regulation of bladder-type AQP in the pelvic skin and negative posttranscriptional regulation of pelvic skin-type AQP provide flexibility in the water regulation mechanisms, which might have contributed to the evolutionary adaptation of anurans to a wide variety of water environments.

Expression of prolactin messenger ribonucleic acid in the mouse gonads during sexual maturation

The pituitary hormone prolactin is known to be produced in various extrapituitary tissues as well. We examined the presence of prolactin mRNA in the mouse gonads by reverse transcription (RT)-PCR and nucleotide sequence analysis. Both ovary and testis were found to express small amounts of mRNA coding prolactin, identical to that in the pituitary gland. Next, we established a sensitive competitive RT-PCR method to estimate the amount of prolactin mRNA and then measured its expression in the gonads during sexual maturation (10-80 days of age). In the ovary, the amount of prolactin mRNA (copies/microg of total RNA) gradually decreased from day 10 to 40, and the lower level was maintained until day 80. Little difference in the amount was observed between the estrous and diestrous groups on day 80. Conversely, the amount of testicular prolactin mRNA gradually increased from day 10 to 80. These results suggest that prolactin is produced in the mouse gonads and that it acts as an autocrine/paracrine factor modulating gonadal functions.

Vitamin A Insufficiency Accelerates the Decrease in the Number of Sperm Induced by an Environmental Disrupter, Bisphenol A, in Neonatal Mice

Abstract Exposure of neonatal mice to an estrogenic endocrine disruptor, bisphenol A, resulted in a malfunction of the testes when the animals became adults. The effect of bisphenol A was cancelled out by concurrent administration of retinol acetate, a naturally occurring metabolite of vitamin A. In contrast, the effect of endocrine disruption became more severe in mice neonatally exposed to bisphenol A and nursed by mothers fed a vitamin A-deficient diet only a few days before and after parturition. These results clearly show that maternal vitamin A is important for relieving in a baby the effect of endocrine disruption caused by environmental xenoestrogens, and suggest that the changes in the content of vitamin A and similar physiological factors in the habitat may be worth considering in studies on environmental disruptors.