Chikashi Tachi

Content and localization of myostatin in mouse skeletal muscles during aging, mechanical unloading and reloading

Changes in myostatin content and localization in mouse skeletal muscles were investigated during aging, hindlimb suspension (HS) and reloading after HS. During aging, the content of myostatin among solubilized proteins in gastrocnemius and plantaris muscles (Gast/Plant) was initially low and increased until their wet weight/body weight ratio reached a peak. It remained unchanged with further aging, although gradual atrophy of the muscles was seen to occur. Also, the myostatin content did not change significantly during HS (up to 14 days) in both Gast/Plant and soleus muscles, though the muscles showed morphological signs of atrophy. However, reloading for 2 days after a 14-day HS caused significant decreases in the myostatin content in both of these muscles. Immunohistochemical observations showed the sarcoplasmic existence of myostatin, the amount of which appeared to decrease after reloading. The results suggest that myostatin plays a part in the processes of muscular growth and loading-induced hypertrophy, but is not involved in either aging-related or unloading-induced muscular atrophy.

Ultrastructural studies on maternal-embryonic cell interaction during experimentally induced implantation of rat blastocysts to the endometrium of the mouse.

Abstract Rat blastocysts collected around noon on Day 5 of pregnancy were transferred to the uterus of the mouse on Day 3 of pseudopregnancy. Out of 430 rat blastocysts transferred, a total of 37 were recovered as xenogeneic implants from the recipient mice killed 36, 48, 52, 58, 72, 96, and 120 hr after transfer. None of the transferred blastocysts was found surviving in the host uterus beyond 96 hr after transfer. Electron microscopic examination of the recovered implants revealed that rat blastocysts can successfully undergo the stages of ovum implantation in the mouse uterus from the early attachment to the initial phase of the trophoblastic invasion of the endometrium. During the late attachment stage, desmosomes (maculae adhaerentes), intermediate junctions (zonulae adhaerentes), and gap junctions (nexuses) were formed xenogeneically between the foreign trophoblast and the uterine epithelial cells of the host. Trophoblast cells of xenogeneic implants were destroyed shortly after the penetration of the basement membrane of the luminal epithelium of the host endometrium, leading to the degeneration and sloughing off of the transferred embryos.

Differential gene expression of β‐1,4‐galactosyltransferases I, II and V during mouse brain development

Since most brain glycoproteins from β‐1,4‐galactosyltransferase (β‐1,4‐GalT) I knockout mice were galactosylated without apparent reduction the gene expression of novel β‐1,4‐GalTs II and V which are involved in N‐linked oligosaccharide biosynthesis in addition to β‐1,4‐GalT I was studied during mouse brain development. Isolation and characterization of β‐1,4‐GalT II and V cDNAs from mouse brains indicates that they are also functioning in the brain. Northern blot analysis revealed that the β‐1,4‐GalT I gene is expressed mainly in mid‐embryonic stages, while the expression level of β‐1,4‐GalT II transcript remains constant and of β‐1,4‐GalT V transcript increases during mouse brain development after birth. In situ hybridization revealed that β‐1,4‐GalT II and V signals are present in most neural cells, with a marked difference between them in the hippocampus of adult mouse brain tissue. The differential gene expression of β‐1,4‐GalTs I, II and V during mouse brain development could affect the differential galactosylation of brain glycoproteins, as revealed by lectin blot analysis.

Possible role for the c-ski gene in the proliferation of myogenic cells in regenerating skeletal muscles of rats.

Skeletal muscle regeneration after injury involves various processes, such as infiltration by inflammatory cells, the proliferation of satellite cells and fusion to myotubes. The c‐ski nuclear protein has been implicated in the control of cell proliferation and/or terminal differentiation in the growth of skeletal muscle. However, there have been no reports concerning the involution of c‐ski in the regeneration of injured skeletal muscle in mammals. A possible role for c‐ski in the proliferation of myogenic cells in rat skeletal muscle during regeneration has been investigated with the assistance of in vitro experiments with L6 skeletal muscle cells. The expression levels of c‐ski mRNA in regenerating tissues increased to approximately threefold that of intact tissues at 2 days after injury and decreased to normal levels at 2 weeks after injury. Many mononuclear cells among the Ski‐positive cells expressed desmin and proliferating cell nuclear antigen, indicating that Ski‐producing cells include the proliferating myogenic cells. The proliferation of L6 cells was significantly retarded by expression of the antisense ski gene. The results of the present study reveal that the c‐ski gene plays an important role in the proliferation of myogenic cells in the regeneration of injured skeletal muscle.

Co-injection of restriction enzyme with foreign DNA into the pronucleus for elevating production efficiencies of transgenic animals

The microinjection method for production of transgenic farm animals requires specialized techniques and results in intolerably low production efficiencies. We investigated whether or not co-injection of foreign DNA constructs with restriction endonuclease into the pronucleus of mouse zygotes would improve the integration frequencies of foreign DNA into the host genome. Two kinds of DNA constructs that have no EcoRI site in their sequences were used for co-microinjection. With reference to the results of experiments in which EcoRI alone was injected at various amounts varying from 10(-9) to 10(-5) U/nucleus, the amount of 5x10(-8) U/nucleus that showed survival rate of 60.6% was used for the co-injection with DNA. Successful transgenesis of co-injected embryos was identified by DpnI-Bal31 digestion method for single embryos and by PCR method for pups born, respectively. The overall efficiency for the integration of foreign DNA in single embryos and live-born pups obtained by the co-injection procedures were 17.9% compared with 9.1% obtained by the injection of DNA alone. The results suggest that co-injection of foreign genes with restriction enzyme may elevate the integration rate of foreign genes into host genomes.

Equine inhibin/activin βA-subunit mRNA is expressed in the endometrial gland, but not in the trophoblast, during pregnancy

The expression of both inhibin α‐ and inhibin/activin βA‐subunit mRNA was examined in equine uteroplacental tissues collected during pregnancy (days 90 to 300). Northern blot analysis revealed that 5 transcripts (7.0, 4.1, 3.4, 2.6, 1.5 kb) of βA‐subunit were present, and the most abundantly expressed transcript was the 1.5 kb one. Relatively high levels of the 1.5 kb transcript were seen in the second trimester of pregnancy compared to what was found in the third trimester. To identify the tissue localization of βA‐subunit mRNA, in situ hybridization was performed, and the positive signal was observed exclusively in the endometrial glands, but not in the fetal placental tissue (trophoblast) at days 150, 210, and 300 of pregnancy. On the other hand, inhibin α‐subunit transcript could not be detected at any stage of pregnancy examined either by Northern blot analysis or in situ hybridization. Although the factor(s) regulating the gene expression of βA‐subunit in this equine tissue is currently unknown, these results suggest that activin, but not inhibin, is predominantly produced in the endometrial glands of the pregnant mare, and thus produced activin may play a paracrine or endocrine role during pregnancy in this species. Mol. Reprod. Dev. 47:363–369, 1997.

Electron microscopic studies of chimeric blastocysts experimentally produced by aggregating blastomeres of rat and mouse embryos

Xenogeneic chimeras between rat and mouse were produced by aggregating embryos at the 8- to 12-cell stages. Of 114 combinations we have made so far, 26 chimeric embryos developed into blastocysts. The origin of each cell in the composite embryos can be identified unambiguously by the ultrastructural appearance of the cytoplasmic inclusions. Both the rat- and the mouse-derived cells differentiated equally well into either ICM or trophoblast cells. However, the mouse-derived cells gave rise to ICM cells more frequently than the rat-derived cells. Furthermore, when the ratderived cells formed trophoblast cells, they were predominantly mural trophoblast cells, while the mouse-derived cells differentiated predominantly into the polar trophoblast cells. Cells of the same species tend to remain as a group in the chimeric blastocysts.

Partial Characterization of Macromolecular Components in Fetal Bovine Serum Required for Development of Mouse Blastocysts Cultured in vitro

Elucidation of the mechanisms underlying implantation of blastocysts in eutherian mammals have been severely hampered by the lack of a suitable system for culture of blastocysts in vitro. Successful culture methods for mouse peri‐implantation embryos in vitro have been described by Hsu (1971, 1973, 1978, 1990), Chen and Hsu (1982) and Naruse et al. (1985), but these methods are too complex for routine experimental purposes. We attempted, therefore, to establish a standard culture method suitable for quantitative analysis of early embryogenesis in the mouse. Our system allows the development of peri‐implantation blastocysts from the time of shedding of the zona to formation of the proamniotic cavity under well defined conditions. Using this system, the macromolecular components in the sera required for the development of periimplantation mouse blastocysts in vitro were partially characterized. Results indicated that substances with molecular weight (MW) of 30 × 103 to 100 × 103 in the serum are capable of inducing the early phase of the trophoblast spreading. Furthermore, serum factors above MW 100 × 103 were found to be essential for the successful differentiation and/or development of ICM and ectoplacental cones.

Comparative analysis of HOXC‐9 gene expression in murine hemochorial and caprine synepitheliochorial placentae by in situ hybridization

Mammalian placentae exhibit wide structural diversity among different species and are formed under intricate interplay between the embryonic trophoblast and the maternal endometrial cells. Increasing evidence in the literature indicates a possible role played by homeobox genes in the complex placental organogenesis. Although the expression of all HOX 9 paralogs has been demonstrated both in highly invasive murine hemochorial placentae and in non‐invasive caprine synepitheliochorial placentae, no reports so far published in the literature described the patterns of gene expression of Hoxc‐9 in the murine nor those of HOXC‐9 in the caprine placenta at cellular levels. We carried out comparative analyses of the location and identity of the cells expressing Hoxc‐9/HOXC‐9 during various stages of placentation in the murine hemochorial and caprine synepitheliochorial placentae by means of in situ hybridization using murine Hoxc‐9 or caprine HOXC‐9 cRNA probe, respectively. The results demonstrated that Hoxc‐9 mRNA was expressed at high levels in giant trophoblast cells of murine placentae on Days 12–19, but not on Day 8. Similar analysis of caprine Day 75 and Day 100 placentae revealed that the binucleate trophoblast cells that penetrate the uterine luminal epithelial cell layer, strongly expressed HOXC‐9 mRNA. Although the functional significance of Hoxc‐9/HOXC‐9 gene expression in trophoblast cells remains to be elucidated, it was suggested that it might play a role in the regulation of invasiveness or endocrine activities in the murine giant trophoblast cells and/or the caprine binucleate trophoblast cells. Anat Rec 259:383–394, 2000.

Molecular cloning of cDNA encoding the c-kit receptor of Shiba goats and a novel alanine insertion specific to goats and sheep in the kinase insert region

The complete open reading frame (ORF) of the c-kit cDNA was cloned from a cerebellar cDNA library of the Shiba goat (Capra hircus var Shiba) with the dominant black-eyed white phenotype. The analysis of the deduced amino acid sequence revealed the presence of a single amino acid insertion (alanine) in the kinase insert (KI) region. While the newly found alanine insertion is not correlated with the coat color phenotype of goats, it appears to be characteristic of the c-kit genes in goats and sheep. Although the biological significance of the insert remains to be investigated, its phylogenetically limited distribution will provide us with a useful and interesting tool to analyze the problems of evolution of sheep and goats in bovidae.