Zoological Society of JapanActa Medica Okayama0289-00034012023Fbxl4 Regulates the Photic Entrainment of Circadian Locomotor Rhythms in the Cricket Gryllus bimaculatus5363ENKazukiTakeuchiGraduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, JapanMiraiMatsukaGraduate School of Natural Science and Technology, Okayama UniversityTsugumichiShinoharaGraduate School of Natural Science and Technology, Okayama UniversityMayukoHamadaUshimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama UniversityYasuakiTomiyamaGraduate School of Natural Science and Technology, Okayama UniversityKenjiTomiokaGraduate School of Natural Science and Technology, Okayama UniversityPhotic entrainment is an essential property of the circadian clock that sets the appropriate timing of daily behavioral and physiological events. However, the molecular mechanisms underlying the entrainment remain largely unknown. In the cricket Gryllus bimaculatus, the immediate early gene c-fosB plays an important role in photic entrainment, followed by a mechanism involving cryptochromes (crys). However, the association between c-fosB expression and crys remains unclear. In the present study, using RNA-sequencing analysis, we found that five Fbxl family genes (Fbxl4, Fbxl5, Fbxl16, Fbxl-like1, and Fbxl-like2) encoding F-box and leucine-rich repeat proteins are likely involved in the mechanism following light-dependent c-fosB induction. RNA interference (RNAi) of c-fosA/B significantly downregulated Fbxls expression, whereas RNAi of the Fbxl genes exerted no effect on c-fosB expression. The Fbxl genes showed rhythmic expression under light-dark cycles (LDs) with higher expression levels in early day (Fbxl16), whole day (Fbxl-like1), or day-to-early night (Fbxl4, Fbxl5, and Fbxl-like2), whereas their expression was reduced in the dark. We then examined the effect of their RNAi on the photic entrainment of the locomotor rhythm and found that RNAi of Fbxl4 either disrupted or significantly delayed the re-entrainment of the locomotor rhythm to shifted LDs. These results suggest that light-induced c-fosB expression stimulates Fbxl4 expression to reset the circadian clock.No potential conflict of interest relevant to this article was reported.Zoological Society of JapanActa Medica Okayama0289-00033952022Constant Light, Pdp1, and Tim Exert Influence on Free-Running Period of Locomotor Rhythms in the Cricket Gryllus bimaculatus459467ENYoshiyukiMoriyamaDepartment of Natural Sciences, Kawasaki Medical School, Kurashiki 701-0192, JapanKazukiTakeuchiGraduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, JapanKenjiTomiokaGraduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, JapanMost insects show circadian rhythms of which the free-running period changes in a light-dependent manner and is generally longer under constant light (LL) than under constant dark conditions in nocturnal animals. However, the mechanism underlying this LL-dependent period change remains unclear. Here, using the cricket Gryllus bimaculatus, we examined the effects of long-term LL exposure on the free-running period of locomotor rhythms. Initially, the free-running period was considerably longer than 24 h but it gradually became shorter during long-term exposure to LL. The initiallengthening and ensuing gradual shortening under long-term LL exposure were observed evenafter unilateral removal of the optic lobe. Thus, these changes in the free-running period could be attributable to a single optic lobe clock. RNA interference (RNAi)-mediated silencing of the clock genes Par domain protein 1 (Pdp1) and timeless (tim) revealed that the treatments eliminated the initial period lengthening by LL without reducing circadian photoreceptor gene expression. However, they did not affect the period shortening during long-term LL exposure. The slopes of the regression line for the period change during long-term LL for Pdp1RNAi-treated and timRNAi-treated crickets were not different from that of the dsDsRed2-treated control. These results suggest that the initial period lengthening after transfer to LL requires tim and Pdp1, while the ensuing period shortening during long-term LL exposure is caused by a mechanism independent of tim and Pdp1. No potential conflict of interest relevant to this article was reported.Zoological Society of JapanActa Medica Okayama0289-00033912022Observing Phylum-Level Metazoan Diversity by Environmental DNA Analysis at the Ushimado Area in the Seto Inland Sea157165ENTakeshiKawashimaNational Institute of GeneticsMasa-akiYoshidaMarine Biological Science Section, Education and Research Center Biological Resources, Faculty of Life and Environmental Science, Shimane UniversityHideyukiMiyazawaNational Institute of GeneticsHiroakiNakanoShimoda Marine Research Center, University of TsukubaNatumiNakanoDepartment of Biology, Nara Medical UniversityTatsuyaSakamotoUshimado Marine Institute, Okayama UniversityMayukoHamadaUshimado Marine Institute, Okayama UniversityThe dynamics of microscopic marine plankton in coastal areas is a fundamental theme in marine biodiversity research, but studies have been limited because the only available methodology was collection of plankton using plankton-nets and microscopic observation. In recent years, environmental DNA (eDNA) analysis has exhibited potential for conducting comprehensive surveys of marine plankton diversity in water at fixed points and depths in the ocean. However, few studies have examined how eDNA analysis reflects the actual distribution and dynamics of organisms in the field, and further investigation is needed to determine whether it can detect distinct differences in plankton density in the field. To address this, we analyzed eDNA in seawater samples collected at 1 km intervals at three depths over a linear distance of approximately 3.0 km in the Seto Inland Sea. The survey area included a location with a high density of Acoela (Praesagittifera naikaiensis). However, the eDNA signal for this was little to none, and its presence would not have been noticed if we did not have this information beforehand. Meanwhile, eDNA analysis enabled us to confirm the presence of a species of Placozoa that was previously undiscovered in the area. In summary, our results suggest that the number of sequence reads generated from eDNA samples in our project was not sufficient to predict the density of a particular species. However, eDNA can be useful for detecting organisms that have been overlooked using other methods.No potential conflict of interest relevant to this article was reported.Zoological Society of JapanActa Medica Okayama0289-00032292005Organ-Specific and Age-Dependent Expression of Insulin-like Growth Factor-I (IGF-I) mRNA Variants: IGF-IA and IB mRNAs in the Mouse10111021ENTakashiOhtsukiMarikoOtsukiYousukeMurakamiTetsuyaMaekawaTakashiYamamotoKojiAkasakaSakaeTakeuchiSumioTakahashiInsulin-like growth factor-I (IGF-I) gene generates several IGF-I mRNA variants by alternative splicing. Two promoters are present in mouse IGF-I gene. Each promoter encodes two IGF-I mRNA variants (IGF-IA and IGF-IB mRNAs). Variants differ by the presence (IGF-IB) or absence (IGF-IA) of a 52-bp insert in the E domain-coding region. Functional differences among IGF-I mRNAs, and regulatory mechanisms for alternative splicing of IGF-I mRNA are not yet known. We analyzed the expression of mouse IGF-IA and IGF-IB mRNAs using SYBR Green real-time RT-PCR. In the liver, IGF-I mRNA expression increased from 10 days of age to 45 days. In the uterus and ovary, IGF-I mRNA expression increased from 21 days of age, and then decreased at 45 days. In the kidney, IGF-I mRNA expression decreased from 10 days of age. IGF-IA mRNA levels were higher than IGF-IB mRNA levels in all organs examined. Estradiol-17 beta (E2) treatment in ovariectomized mice increased uterine IGF-IA and IGF-IB mRNA levels from 3 hr after injection, and highest levels for both mRNAs were detected at 6 hr, and relative increase was greater for IGF-IB mRNA than for IGF-IA mRNA. These results suggest that expression of IGF-I mRNA variants is regulated in organ-specific and age-dependent manners, and estrogen is involved in the change of IGF-I mRNA variant expression.No potential conflict of interest relevant to this article was reported.Zoological Society of JapanActa Medica Okayama0289-00032292005Interleukin-18 (IL-18) mRNA Expression and Localization of IL-18 mRNA-Expressing Cells in the Mouse Uterus10031010ENKenjiKusumotoYousukeMurakamiMarikoOtsukiMunetoshiKanayamaSakaeTakeuchiSumioTakahashiInterleukin-18 (IL-18) belongs to the interleukin-1 family and was identified as an interferon gamma inducing factor. We investigated IL-18 mRNA-expressing cells in the mouse uterus. By RNase protection assay, IL-18 mRNA and a subunit of IL-18 receptor mRNA were detected in the uterus. In the uterus, IL-18 mRNA levels increased during sexual maturation. In situ hybridization analysis demonstrated IL-18 mRNA-expressing cells in the mouse uterus of different ages. At 21 days of age, IL-18 mRNA-expressing cells were detected in the luminal epithelial cells and stromal cells although the IL-18 mRNA signal was weak. At 42 days of age, IL-18 mRNA signal was mainly detected in the stromal cells located near the myometrium, and in some of the luminal and glandular epithelial cells. In the uterus of 63-day-old adult mice, a strong hybridization signal for IL-18 mRNA was detected at estrus, but was weak at diestrus. IL-18 mRNA was mainly detected in the glandular epithelial cells and stromal cells. The effect of estradiol-17 beta (E-2) on IL-18 mRNA-expressing cells in the uterus was examined in ovariectomized mice. In oil-treated mice IL-18 mRNA signal was localized in luminal epithelial cells and stromal cells, while in E-2-treated mice IL-18 mRNA signal was localized in stromal cells alone. These results suggest that the mouse uterus has an IL-18 system, and IL-18 exerts a physiological role within the uterus in a paracrine manner, and that IL-18 gene expression is regulated by estrogen.No potential conflict of interest relevant to this article was reported.Zoological Society of JapanActa Medica Okayama0289-000323112006Characterization of Mouse Tissue Kallikrein 5963968ENSanathRajapakseKatsuekiOgiwaraNorikoYamanoAtsushiKimuraKensakuHirataSumioTakahashiTakayukiTakahashiMouse tissue kallikreins (Klks) are members of a large, multigene family consisting of 37 genes, 26 of which can code for functional proteins. Mouse tissue kallikrein 5 (KIk5) has long been thought to be one of these functional genes, but the gene product, mK5, has not been isolated and characterized. In the present study, we prepared active recombinant mK5 using an Escherichia coli expression system, followed by column chromatography. We then determined the biochemical and enzymatic properties of purified mK5. mK5 had trypsin-like activity for Arg at the P1 position, and its activity was inhibited by typical serine protease inhibitors. mK5 degraded gelatin, fibronectin, collagen type IV, high-molecular-weight kininogen, and insulin-like growth factor binding protein-3. Our data suggest that mK5 may be implicated in the process of extracellular matrix remodeling.No potential conflict of interest relevant to this article was reported.Zoological Society of JapanActa Medica Okayama0289-00032432007Alternative Leader-Exon Usage in Mouse IGF-I mRNA Variants: Class 1 and Class 2 IGF-I mRNAs241247ENTakashiOhtsukiMarikoOtsukiYousukeMurakamiKensakuHirataSakaeTakeuchiSumioTakahashiThe mouse IGF-I gene contains six exons, and exon 1 and exon 2 gene are considered to be leader exons. The regulatory mechanism of alternative usage of the leader exons is unclear in mice. The present study, was aimed at clarifying changes in class 1 (derived from exon 1) and class 2 (derived from exon 2) IGF-I mRNA expression in mice under various conditions. Both class 1 and class 2 IGF-I mRNAs were expressed in the mouse uterus, liver and kidney, and class 1 IGF-I mRNA was the major transcript in all organs studied. In the uterus, both class 1 and class 2 IGF-I mRNA expression changed markedly during the estrous cycle, with the highest level at proestrus, but in the liver and kidney there were no significant changes in IGF-I mRNA expression during the estrous cycle. Estrogen treatment increased both class 1 and class 2 IGF-I mRNA levels in the uterus of ovariectomized mice, but class 1 mRNA expression increased more in response to estrogen treatment than class 2 mRNA expression. These findings suggest that estrogen stimulates IGF-I gene expression in, uterine cells, and that a promoter involved in transcription of class 1 IGF-I mRNA is more responsive to estrogen. In conclusion, the present study revealed that two leader exons of mouse IGF-I gene are used in the uterus, liver and kidney. IGF-I mRNA levels of both classes changed during the estrous cycle in the uterus, but not in the liver or kidney. Estrogen increased IGF-I mRNA levels of both classes in the uterus.No potential conflict of interest relevant to this article was reported.Zoological Society of JapanActa Medica Okayama0289-00032622009Estradiol, Progesterone, and Transforming Growth Factor ƒ¿ Regulate Insulin-Like Growth Factor Binding Protein-3 (IGFBP3) Expression in Mouse Endometrial Cells131138ENTetsuyaMaekawaSakaeTakeuchiMunetoshiKanayamaSumioTakahashiInsulin-like growth factor 1 (IGF1) Is Involved in the proliferation of mouse and rat endometrial cells in a paracrine or autocrine manner. Insulin-like growth factor binding protein-3 (IGFBP3) modulates actions of IGFs directly or indirectly. The present study aimed to determine whether IGFBP3 is Involved In the regulation of proliferation of mouse endometrial cells. Mouse endometrial epithelial cells and stromal cells were isolated, and cultured In a serum free medium. IGF1 stimulated DNA synthesis by endometrial epithelial and stromal cells, and IGFBP3 Inhibited IGF1-induced DNA synthesis. Estradiol-17 beta (E2) decreased the Igfbp3 mRNA level in endometrial stromal cells, whereas It Increased the Igf1 mRNA level. Transforming growth factor alpha (TGF alpha) significantly decreased IGFBP3 expression at both the mRNA and secreted protein levels in endometrial stromal cells. Progesterone (134) did not affect the E2-induced down-regulation of Igfbp3 mRNA expression in endometrial stromal cells, although P4 alone increased Igfbp3 mRNA levels. The present findings suggest that in mouse endometrial stromal cells E2 enhances IGF1 action through enhancement of IGF1 synthesis and reduction of IGFBP3 synthesis, and that TGF alpha affects IGF1 actions through modulation of IGFBP3 levels.No potential conflict of interest relevant to this article was reported.Zoological Society of JapanActa Medica Okayama0289-00032462007Effects of Unilateral Compound-Eye Removal on the Photoperiodic Responses of Nymphal Development in the Cricket Modicogryllus siamensis604610ENTomoakiSakamotoKenjiTomiokaThe cricket, Modicogryllus siamensis, shows clear photoperiodic responses at 25 degrees C in nymphal development. Under long-day conditions (LD16:8), nymphs became adults about 50 days after hatching, while under short-day conditions (LD8:16) the duration of nymphal stage extended to more than 130 days. Under constant dark conditions, two developmental patterns were observed: about 60% of crickets became adults slightly slower than under the long-day conditions, and the rest at later than 100 days after hatching, like those under the short-day conditions. When the compound eye was unilaterally removed on the 2nd day of hatching, an increase of molting and an extension of the nymphal period were observed under the long-day conditions, while under the short-day conditions, some crickets developed faster and others slower than intact crickets. These results suggest that this cricket receives photoperiodic information through the compound eye, that a pair of the compound eyes is required for a complete photoperiodic response, and that interaction between bilateral circadian clocks may be also involved in the response.No potential conflict of interest relevant to this article was reported.Zoological Society of JapanActa Medica Okayama0289-000325112008Behavioral Dissection of the Drosophila Circadian Multioscillator System that Regulates Locomotor Rhythms11461155ENYujiroUmezakiKenjiTomiokaThe fruit fly, Drosophila melanogaster, shows a bimodal circadian activity rhythm with peaks around light-on and before light-off. This rhythm is driven by seven groups of so-called clock neurons in the brain. To dissect the multioscillatory nature of the Drosophila clock system, the process of reentrainment to a reversed light cycle was examined by using wild-type flies and cry(b) mutant flies that carry a strong loss-of-function mutation in cryptochrome (cry) gene. The wild-type flies showed that the morning peak dissociated into two components, while a substantial fraction of cry(b) flies exhibited dissociation of the evening peak into two components that shifted in different directions. When the temperature cycle was given in constant darkness in such a manner that the thermophase corresponded to the previous night phase, the morning peak also split into two components in wild-type flies. These results suggest that both morning and evening peaks are driven by two separate oscillators that have different entrainability to light and temperature cycles. Examination of the process of reentrainment to a reversed LD in mutant flies that lack some of the four known circadian photoreceptors (compound eyes, ocelli, CRYPTOCHROME [CRY], and Hofbauer-Buchner [H-B] eyelets) revealed that these four photoreceptors play different roles in photic entrainment of the four putative oscillators.No potential conflict of interest relevant to this article was reported.Zoological Society of JapanActa Medica Okayama0289-00031541998Insulin-Like Growth Factor-I and Its Receptor in Mouse Pituitary Glands573579ENJunichiHondaSakaeTakeuchiHiroshiFukumachiSumioTakahashiInsulin-like growth factor-I (IGF-I) is produced in the liver and other peripheral tissues in response to growth hormone (GH) stimuli. IGF-I regulates diverse physiological functions in an autocrine and/or paracrine manner. IGF-I and IGF-I receptor (type-I receptor) are expressed in human and rat pituitary glands. However, the cell types of IGF-I-expressing cells and target cells of IGF-I in the pituitary glands are not known. The present study was aimed to identify the cell types of IGF-I-expressing cells and of its type-I receptor-expressing cells in mouse pituitary glands. In the mouse pituitary glands, IGF-I mRNA and IGF-I receptor mRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR). IGF-I-expressing cells and its receptor-expressing cells were detected by non-radioisotopic in situ hybridization using mouse IGF-I cDNA and IGF-I receptor cDNA probes, and their cell types were immunocytochemically determined using antibodies raised against pituitary hormones. We found that somatotrophs expressed both IGF-I and IGF-I receptors, and some of corticotrophs expressed IGF-I receptors. Co-localization of IGF-I and GH in the same cultured pituitary cells was observed by dual-labelling immunocytochemistry. The present study demonstrated that pituitary IGF-I produced in somatotrophs regulated functions of somatotrophs and corticotrophs in an autocrine and/or paracrine manner.No potential conflict of interest relevant to this article was reported.Zoological Society of JapanActa Medica Okayama0289-00031541998Augmentation of Prolactin Release by ƒ¿-Melanocyte Stimulating Hormone Is Possibly Mediated by Melanocortin 3-Receptors in the Mouse Anterior Pituitary Cells567572ENYoshiakiMorookaSouichiOomizuSakaeTakeuchiSumioTakahashiSuckling- and estrogen-induced prolactin release from the anterior pituitary is mediated by alpha-melanocyte stimulating hormone (a-MSH) secreted by the intermediate lobe of the pituitary in the rat. Melanocortin 5-receptors are expressed in the anterior pituitary and probably mediate the alpha-MSH function. In contrast, the mouse anterior pituitary does not express the receptor. To examine whether or not alpha-MSH regulates prolactin release in mice, we performed cell immunoblot assay using anterior pituitary cells from adult female mice. We found that alpha-MSH acted on mammotrophs (prolactin-secreting cells) and stimulated prolactin release in a dose dependent manner. A series of RT-PCR using oligonucleotide primer pairs specific for each subtypes of melanocortin receptors revealed that the melanocortin 3-receptor is the sole receptor expressed in the mouse anterior pituitary. These results suggest that alpha-MSH-induced prolactin release is mediated by melanocortin 3-receptors in female mice.No potential conflict of interest relevant to this article was reported.Zoological Society of JapanActa Medica Okayama0289-00031752000Epidermal Growth Factor Stimulates Proliferation of Mouse Uterine Epithelial Cells in Primary Culture661666ENMasahiroShiragaNorikoKomatsuKiyoshiTeshigawaraAkinobuOkadaSakaeTakeuchiHiroshiFukamachiSumioTakahashiEpidermal growth factor (EGF) is one of growth factors that are thought to mediate the stimulatory effects of estrogen on the proliferation of uterine epithelial cells. The present study was attempted to obtain direct evidence for the mitogenic effects of EGF on uterine epithelial cells, and to prove that EGF and EGF receptors are expressed in these cells. Mouse uterine epithelial cells were isolated from immature female mice and cultured with or without EGF for 5 days. EGF (1 to 100 ng/ml) significantly increased the number of uterine epithelial cells, and the maximal growth (141.9+/-8.3% of controls) was obtained at a dose of 10 ng/ml. In addition, EGF (0.1 to 100 ng/ml) increased the number of DNA-synthesizing cells immunocytochemically detected by bromodeoxyuridine uptake to the nucleus. Northern blot analysis revealed that the uterine epithelial cells expressed both EGF mRNA (4.7 kb) and EGF receptor mRNAs (10.5, 6.6, and 2.7 kb) These results suggest that the proliferation of uterine epithelial cells is regulated by the paracrine and/ or autocrine action of EGF. Our previous study demonstrated the mitogenic effect of IGF-I on uterine epithelial cells. To examine whether the EGF- and IGF-I signaling act at the same level in the regulation of the proliferation of uterine epithelial cells, the cultured cells were simultaneously treated with IGF-I and EGF. IGF-I was found to additively stimulate the mitogenic effects of EGF, suggesting that the EGF-induced growth of uterine epithelial cells is distinct from IGF-l-induced growth.No potential conflict of interest relevant to this article was reported.Zoological Society of JapanActa Medica Okayama0289-00031972002Induction of mammotroph development by a combination of epidermal growth factor, insulin, and estradiol-17ƒÀ in rat pituitary tumor GH3 cells789795ENTomoshiKakeyaSakaeTakeuchiSumioTakahashiSeveral reports have indicated that prolactin-secreting cells (PRL cells) are generated from growth hormone-secreting cells (GH cells). We have shown that treatment with a combination of epidermal growth factor (EGF), insulin, and estradiol-17beta (E-2) induces the appearance of PRL cells in pituitary tumor GH3 cells. The aim of the present study was to clarify the involvement of mitosis in the cytogenesis of PRL cells in rat pituitary and GH3 cells. The effects of the treatment with EGF, insulin and E-2 on DNA-replication were studied by detecting the uptake of bromodeoxyuridine (BrdU) into the nucleus. In cultured rat pituitary cells, BrdU-labeled PRL cells were observed irrespective of the hormone treatment. In GH3 cells, BrdU-Iabeled GH cells and mammosomatotrophs (MS cells) were detected; BrdU-labeled PRL cells were not detected, however, when GH3 cells were treated with BrdU for 3 hr and then immediately examined for BrdU-labeling. BrdU-Iabeled PRL cells were found only when GH3 cells treated with BrdU were allowed to grow for another 3 days. This finding suggests that during the additional 3-day culture, BrdU-labeled PRL cells were generated from BrdU-Iabeled cells other than PRL cells. These results indicate that PRL cells are transdifferentiated from GH cells or VIS cells in GH3 cells by a combined treatment with EGF, insulin and E-2, while PRL cells in rat pituitaries are able to proliferate in response to the hormone treatment. Thus, there may be two pathways for cytogenesis of PRL cells the transdifferentiation of GH cells or VIS cells, and a self-duplication of PRL cells.No potential conflict of interest relevant to this article was reported.Zoological Society of JapanActa Medica Okayama0289-00032012003Gene Expression and the Physiological Role of Transforming Growth Factor-ƒ¿ in the Mouse Pituitary8389ENSeemaSharmaSouichiOomizuTomoshiKakeyaTohruMasuiSakaeTakeuchiSumioTakahashiTransforming growth factor-alpha (TGF-alpha), a member of the epidermal growth factor (EGF) family, is produced within the mouse anterior pituitaries. However, the cell types of TGF-alpha-expressing cells and the physiological roles of TGF-a within mouse pituitary glands remain unclear. The aim of the present study was to localize TGF-alpha mRNA-expressing cells, and to clarify the involvement of TGF-alpha in estrogen-induced DNA replication in mouse anterior pituitary cells. Northern blot analysis demonstrated TGF-alpha mRNA expression in adult male and female mouse anterior pituitaries. In situ hybridization analysis of the pituitaries in these mice showed that TGF-alpha mRNA-expressing cells in the anterior pituitary are round, oval, and medium-sized. TGF-alpha mRNA was colocalized in most of the growth hormone (GH) mRNA-expressing cells, while only some of the prolactin (PRL) mRNA-expressing cells. DNA replication in the anterior pituitary cells was detected by monitoring the cellular uptake of a thymidine analogue, bromodeoxyuridine (BrdU) in a primary serum-free culture system. Estradiol-17beta (E2) and TGF-alpha treatment increased the number of BrdU-labelled mammotrophs, indicating that E2 and TGF-alpha treatment stimulates the DNA replication in mammotrophs. Immunoneutralization of TGF-alpha with anti-TGF-alpha-antibodies nullified the E2-induced increase in DNA replication. RT-PCR analysis of TGF-alpha mRNA expression in ovariectomized female mice revealed that E2 increases TGF-alpha mRNA levels. These results indicate that the TGF-alpha produced primarily in the somatotrophs mediates the stimulatory effects of estrogen on the DNA replication of pituitary cells in a paracrine or autocrine manner.No potential conflict of interest relevant to this article was reported.Zoological Society of JapanActa Medica Okayama0289-00032052003Epidermal Growth Factor and Transforming Growth Factor-ƒ¿ Stimulate the Proliferation of Mouse Uterine Stromal Cells639645ENNorikoKomatsuTetsuyaMaekawaSakaeTakeuchiSumioTakahashiGrowth factors produced in the uterine endometrium are considered to be involved in the proliferation of the mouse uterine stromal cells induced by estradiol-17beta (E-2) and progesterone (P). The effect of epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha), one of EGF-related growth factors, on the proliferation of mouse uterine stromal cells was studied in a serum-free culture. The growth of the uterine stromal cells was measured by MTT assay. EGF was found to increase the number of uterine stromal cells in a dose-dependent manner. The DNA-replicating cells were investigated using the immunocytochemical detection of bromodeoxyuridine (BrdU)-labeled cells. EGF and TGF-alpha increased the percentage of BrdU-Iabeled cells in a dose-dependent manner. Administration of the combination of E-2 (10(-9) M) and P (10(-7) M) for 2 days increased the percentage of BrdU-Iabeled cells 2.3-fold. The stimulatory effect of EGF, TGF-a and the combination of E2 and P on DNA replication in the uterine stromal cells was repressed by RG-13022 (10(-5) M, the inhibitor of the EGF receptor tyrosine kinase). RT-PCR analysis of EGF-receptor-, TGF-alpha, and EGF-mRNA was carried,out in the cultured uterine stromal cells, and revealed the expression of those mRNAs. These data supported the hypothesis that uterine endometrial stromal growth induced by sex steroids required the EGF family of ligands such as EGF and TGF-alpha, both produced in the stromal cells, acting for DNA synthesis through EGF receptors.No potential conflict of interest relevant to this article was reported.Zoological Society of JapanActa Medica Okayama0289-00032162004Intrapituitary Regulatory System of Proliferation of Mammotrophs in the Pituitary Gland601611ENSumioTakahashiAnterior pituitary cells produce growth factors plus cytokines and their receptors. Although some of these pituitary growth factors and cytokines are known to be involved in the control of cell differentiation, proliferation and hormone production in the pituitary gland, their physiological roles remain unknown. Lots of evidence indicates that they are involved in the regulation of prolactin-secreting mammotroph cell proliferation. The regulation of mammotroph functions is a suitable system for understanding the intrapituitary regulatory system operated by growth factors and cytokines, since mammotrophs are the most actively proliferating cells in female pituitary glands. This review discusses the possible intrapituitary regulation of mammotroph differentiation and proliferation in rat and mouse pituitaries.No potential conflict of interest relevant to this article was reported.Zoological Society of JapanActa Medica Okayama0289-000320112003Phase Shifts of the Circadian Locomotor Rhythm Induced by Pigment-Dispersing Factor in the Cricket Gryllus bimaculatus13471354ENMuniyandiSingaravelYukoFujisawaMikiHisadaASMSaifullahKenjiTomiokaPigment-dispersing factors (PDFs) are octadeca-peptides widely distributed in insect optic lobes and brain. In this study, we have purified PDF and determined its amino acid sequence in the cricket Gryllus bimaculatus. Its primary structure was NSEIINSLLGLPKVLNDA-NH2, homologous to other PDH family members so far reported. When injected into the optic lobe of experimentally blinded adult male crickets, Gryllus-PDF induced phase shifts in their activity rhythms in a phase dependent and dose dependent manner. The resulted phase response curve (PRC) showed delays during the late subjective night to early subjective day and advances during the mid subjective day to mid subjective night. The PRC was different in shape from those for light, serotonin and temperature. These results suggest that PDF plays a role in phase regulation of the circadian clock through a separate pathway from those of other known phase regulating agents.No potential conflict of interest relevant to this article was reported.Zoological Society of JapanActa Medica Okayama0289-000321122004Circadian Organization in Hemimetabolous Insects11531162ENKenjiTomiokaSalaheldinAbdelsalamThe circadian system of hemimetabolous insects is reviewed in respect to the locus of the circadian clock and multioscillatory organization. Because of relatively easy access to the nervous system, the neuronal organization of the clock system in hemimetabolous insects has been studied, yielding identification of the compound eye as the major photoreceptor for entrainment and the optic lobe for the circadian clock locus. The clock site within the optic lobe is inconsistent among reported species; in cockroaches the lobula was previously thought to be a most likely clock locus but accessory medulla is recently stressed to be a clock center, while more distal part of the optic lobe including the lamina and the outer medulla area for the cricket. Identification of the clock cells needs further critical studies. Although each optic lobe clock seems functionally identical, in respect to photic entrainment and generation of the rhythm, the bilaterally paired clocks form a functional unit. They interact to produce a stable time structure within individual insects by exchanging photic and temporal information through neural pathways, in which
serotonin and pigment-dispersing factor (PDF) are involved as chemical messengers. The mutual interaction also plays an important role in seasonal adaptation of the rhythm.No potential conflict of interest relevant to this article was reported.