Masatoshi Komiyama

Origin and development of the epicardium in the mouse embryo

SummaryThe formation of the epicardium was investigated in the mouse embryo using scanning electron microscopy (SEM) in order to establish a three-dimensional perspective concerning epicardial development in mammals. The epicardium first appears as aggregates of cells scattered on the caudal surface of the ventricle and atria where these regions face the septum transversum in a 9-day-old embryo. These aggregated cells seem to have originated from the mesothelial projections extending from the surface of the septum transversum. Then, the cells of each aggregate flatten, subsequently fusing with each other to form a continuous sheet of epicardium. The fusion of aggregates proceeds in a cranial direction. Finally, the bulbus cordis and truncus arteriosus become invested by migrating cells at the cranial end of the epicardial sheet about 11 days after fertilization. The present observations are discussed in comparison with those made previously in avian embryos.

Spatial relationship of nebulin relative to other myofibrillar proteins during myogenesis in embryonic chick skeletal muscle cellsin vitro

SummaryThe developmental expression of nebulin was studied in embryonic chick skeletal muscle cellsin vitro by means of immunofluorescence microscopy. Initially nebulin appeared homogeneously or in a punctate form in the cytoplasm, and then it was assembled into I-Z-I-like complexes containing actin andα-actinin but not myosin and connectin (titin). Striated patterns of nebulin (‘singlets’) in myofibrils appeared simultaneously with those ofα-actinin (Z-bands), myosin (A-bands) and connectin (‘doublets’), but earlier than those of actin. After actin striations were formed as myofibrils matured, each nebulin band started to exhibit ‘droplets’. The delayed development of nebulin compared to the I-Z-I brush formation and the myofibril maturation seems to indicate that this giant myofibrillar protein is unnecessary for both the initial (formation of I-Z-I-like structures) and the subsequent (regular alignment of myofibrils) phases of myofibrillogenesis.

Assembly of connectin (titin) in relation to myosin and α-actinin in cultured cardiac myocytes

SummaryBy using polyclonal and monoclonal antibodies against connectin (titin) which stain the A-I junctional area and the A-band domain (polyclonal anti-connectin and monoclonal 4C9) and the I-band domain (monoclonal SM1), the developmental relationship of this elastic protein with sarcomeric proteins, especially myosin andα-actinin, was examined in embryonic chick cardiac myocytesin vitro under fluorescence microscopy. During premyofibril stages, I-Z-I proteins were detected first (α-actinin dots and diffuse actin [phalloidin and anti-troponin C] staining), and later in these areas connectin and myosin dots appeared with nearly identical distribution. Somewhat later, phalloidin-positive nonstriated fibrils were observed in a straight course. They were always reactive with antibodies against a-actinin and troponin C, but unreactive or only weakly reactive with anticonnectin and anti-myosin. Initially,α-actinin dots were aligned along these fibrils but did not form striations. As they aggregated to form Z-bands, connectin and myosin started to exhibit typical striations (‘doublets’ and A-bands, respectively). No difference in the staining pattern was observed with two kinds of monoclonal antibodies against different domains of connectin filaments (4C9 and SM1) at early phases. As myosin staining began to show clear A-bands, connectin epitopes became arranged in polarized positions. We conclude that primitive I-Z-I complexes appear prior to the assembly of connectin and myosin filaments and then connectin filaments, developing intimately and coordinately with myosin, become associated with theα-actinin lines. Thus it appears that the putative elastic protein connectin plays some role in integrating myosin filaments with the preexisting I-Z-I brushes. The occasional absence of connectin and A-bands between two Z-bands, beyond both of which clear sarcomeres have been formed, indicates that connectin is not a preformed scaffold of myofibrils on which sarcomeric proteins accumulate.

Effect of Exposure to High Isoflavone-Containing Diets on Prenatal and Postnatal Offspring Mice

Isoflavone (IF), a type of phytoestrogen, has multiple beneficial effects, but too much phytoestrogen can have adverse effects on offspring. To examine whether chronic exposure to high IF has adverse effects on reproductive development, mice offspring were exposed to IF through dietary administration to dams during pregnancy and lactation and to the offspring directly after weaning until sacrifice. In male offspring, there was no difference between the IF group and controls; however, in female offspring in the IF group, remarkably earlier puberty and induction of multioocyte follicles on postnatal day (PND) 21 were observed. Gene expression levels of estrogen receptor β decreased in the ovary and vagina on PND 21. These results suggest that chronic exposure to higher than normal levels of IF induces alterations in the reproductive development of female mice through an estrogenic effect.

Exchangeability of actin in cardiac myocytes and fibroblasts as determined by fluorescence photobleaching recovery.

Rhodamine (Rho)-labeled muscle and non-muscle actins were microinjected into cultured embryonic chicken cardiac myocytes and fibroblasts. After incorporation of the fluorescent actin analog into cellular structures, small areas of labeled structures were photobleached with a laser pulse, and fluorescence recovery (FR) was measured to determine the exchangeability of isoactins in these structures. With both Rho-muscle and Rho-non-muscle actins, the FR rate in any part of stress fibers was consistently faster than that observed in any part of myofibrils. Thus, although non-striated (proximal and terminal) portions of nascent myofibrils are similar in appearance and composition to stress fibers, our data clearly revealed differences in actin stability between these two structures. Further, although cardiomyocytes were incapable of discriminating between the incorporation of muscle and non-muscle actin isoforms into myofibrils, FR after photobleaching of Rho-muscle actin was faster than that of Rho-non-muscle actin in immature non-striated portions. This indicates that actin molecules in cardiac myofibrils cannot be readily exchanged by heterotypic non-muscle actin. Fluorescently labeled actin incorporated into non-striated (proximal and terminal) portions of myofibrils and terminal portions of stress fibers was found to be more stable than alpha-actinin. The relative stability of actin could facilitate the formation of nascent Z-bands of myofibrils and the reorganization of stress fibers at these portions.

Identification and Characterization of Novel and Unknown Mouse Epididymis-Specific Genes by Complementary DNA Microarray Technology

Abstract To examine epididymal function, we attempted to identify highly expressed genes in mouse epididymis using a cDNA microarray containing PCR products amplified from a mouse epididymal cDNA library. We isolated one novel and four known genes—lymphocyte cytosolic protein 1 (Lcp1), complement subcomponents C1r/C1s, Uegf protein, and bone morphogenetic protein and zona pellucida-like domains 1 (Cuzd1), transmembrane epididymal protein 1 (Teddm1), and whey acidic protein 4-disulfide core domain 16 (Wfdc16)—with unknown functions in the epididymis. The novel gene, designatedSerpina1f(serine peptidase inhibitor [SERPIN], clade A, member 1f), harbors an open reading frame of 1 233 bp encoding a putative protein of 411 amino acids, including a SERPIN domain. These five genes were predominantly expressed in the epididymis as compared to other organs. In situ hybridization analysis revealed their epididymal region-specific expression patterns. Real-time RT-PCR analysis revealed a significant increase in mRNA expression of these genes around puberty. Castration decreased their expression, except forLcp1. Testosterone (T) restored these reduced expressions, except forTeddm1; however, this restoration was not observed with 17 beta-estradiol (E2). Administration of T and E2 combination recovered theSerpina1fmRNA concentration; this recovery was also observed with T alone. However, the recovery ofCuzd1andWfdc16mRNA concentrations was inadequate. Neonatal diethylstilbestrol treatment suppressed theCuzd1,Wfdc16, andSerpina1fmRNA expression in the epididymis of 8-week-old mice; this was not observed with E2. These results suggest that our microarray system can provide a novel insight into the epididymal function on a molecular basis, and the five genes might play important roles in the epididymis.

CADMIUM EXPOSURE INCREASES SUSCEPTIBILITY TO TESTICULAR AUTOIMMUNITY IN MICE

Cadmium, one of various environmental toxicants, is known to suppress systemic immunity and to injure the testicular capillary endothelia with resultant necrosis of testicular tissues in mice and rats treated with high doses. Recently, it also became evident that cadmium can affect the integrity of the blood–testis barrier (BTB), the endocrine function of Leydig cells, apoptosis of germ cells and systemic immunity, even on treatment with a low dose that does not induce spermatogenic disturbance. Experimental autoimmune orchitis (EAO), i.e., an organ‐specific autoimmunity of the testis, can be induced by repeated immunization with testicular antigens, and its pathology is characterized by lymphocytic inflammation and spermatogenic disturbance. In the present study, we investigated the morphological and functional changes of testes in mice treated with a low dose of cadmium chloride (CdCl2) and also examined its toxicity as to susceptibility to EAO. The results showed that exposure to 3 mg CdCl2 kg−1 body weight did not affect the spermatogenic state. However, the BTB at the tubuli recti and the rete testis, but not the seminiferous tubules, was slightly weakened, and intra‐testicular mRNA expression of interleukin (IL)‐6, tumor necrosis factor‐α and IL‐1β was significantly increased by the CdCl2 treatment. Furthermore, immunization with testicular antigens after the CdCl2 exposure significantly augmented the EAO severity. Therefore, exposure to a low dose of CdCl2 induces no significant disturbance of spermatogenesis, however, it does change the immunological microcircumstances in the testis, resulting in increased susceptibility to testicular autoimmunity. Copyright

Effects on the local immunity in the testis by exposure to di-(2-ethylhexyl) phthalate (DEHP) in mice.

Exposure to di-(2-ethylhexyl) phthalate (DEHP) has been reported to induce spermatogenic disturbance through oxidant stress and affect the immune system as an adjuvant. However, the effect of DEHP on the testicular immune microenvironment has not yet been investigated. In the present study, we examined the testicular immune microenvironment after exposure to doses of DEHP, previously identified as no-observed-adverse-effect levels. Adult male mice were administered food containing 0%, 0.01% or 0.1% DEHP and then testes were analyzed. The results showed that a slight but significant spermatogenic disturbance appeared in the 0.1% DEHP group but not in the 0.01% DEHP group at 8 weeks. It was also demonstrated that lymphocytes and F4/80- and MHC class II- positive cells were significantly increased with the elevation of IL-10 and IFN-γ mRNA expressions in the testes of not only the 0.1% DEHP group but also the 0.01% DEHP group at 8 weeks. Histochemical analyses involving horseradish peroxidase (HRP) as a tracer showed that a little blood-borne HRP had infiltrated into the lumen of a few seminiferous tubules beyond the blood-testis-barrier in both the 0.1% and 0.01% DEHP groups at 8 weeks. This indicates that a dose of DEHP that has little effects on spermatogenesis can change the testicular immune microenvironment with functional damage of the blood-testis barrier.

Differentiation of muscle-specific proteins in chicken somites as studied by immunofluorescence microscopy

Abstract Fluorescence microscopy of chicken cervical somites revealed that muscle-specific proteins began to appear at stage 11 (Hamburger and Hamilton numbering), and the onset of the expression of all the proteins examined in the present study had occurred by stage 17. Muscle proteins were classified into six groups according to the stage of their appearance. Since all these proteins were expressed before emergence of nerve fibers in myotomes, switching-on of their synthesis does not seem to require neuronal influence. However, since isoproteins other than adult muscle types disappeared and diversification of muscle fiber types occurred coordinately with the clustering of acetylcholine receptors in cervical muscles, switching-off of the synthesis of the nonadult isoforms might have been accelerated by the formation of functional neuromuscular junctions. The absence of nebulin and C-protein in early stages seems to indicate that these proteins are not required for the initial assembly of myofilaments and/or myofibrils. Further, this absence might be considered to facilitate exchangeabilities of proteins in nascent myofibrils, thereby changing the isoforms to adult types.

Analysis of toxicogenomic response to endocrine disruptors in the mouse testis

In order to evaluate the feasibility of cDNA microarrays for the risk assessment of endocrine disruptors (EDs), alteration of gene expression profiles was analyzed in adult mouse testes after neonatal exposure to diethylstilbestrol (DES), bisphenol A (BPA) or genistein (Gen), using cDNA microarrays. Analysis with Mouse GEM I revealed that the expression levels of 34, 38 and 12 genes were changed in testes of DES-, Gen- and BPA-treated 12-week old mice, respectively. Gene expression profiles were very similar between DES- and Gen-treated mice, but that of the BPA-treated mice was quite different. These results suggest that gene expression profiles might be feasible for the grouping of EDs in terms of their effects. Further, it is suggested that mechanisms of BPA action may be different from those of DES and Gen, although they are all estrogen-like compounds. Next, we investigated the transition of the expression profiles in testes of DES-treated mice using in-house cDNA microarrays. Many of the genes affected by DES were up- or down-regulated at restricted periods, but some genes were continuously up-regulated or shifted from the down-regulated state to the up-regulated state. These results suggest that the transition patterns may represent the gene expression cascades that were affected by EDs. We propose that the cDNA microarray is a useful tool, which provides us a bird’s eye view of the global effects of EDs on gene expression.