森 千里

Germ cells synthesize large amounts of HSP70-2 protein during the meiotic phase of spermatogenesis. This developmentally regulated expression of HSP70-2 contrasts with the constitutive or inducible expression of other 70-kDa heat shock proteins (HSP70s). To better understand the genetic regulation of Hsp70-2, we used mRNA primer-extension, reverse transcriptase PCR (RT-PCR), and cDNA sequencing to determine that transcription began as far as 353 bp upstream of the start codon. We also identified a previously unrecognized 239-bp intron which is spliced out of the pre-mRNA transcript to leave a 114 nt 5'-untranslated region. Transgenic mice were then produced to delimit the upstream regulatory region required for developmental expression of Hsp70-2 during spermatogenesis. Results with multiple lines of transgenic mice containing promoter-reporter transgenes with varying lengths of Hsp70-2 sequence indicate that promoter sequences up to 640 bp upstream of the start codon and 287 bp upstream of the transcription start site are required for Hsp70-2/lacZ expression in spermatocytes. Histochemical detection of transgene p-galactosidase activity was coincident with immunohistochemical detection of HSP70-2 protein, both in the fist wave of spermatogenesis in juvenile mice and in ongoing spermatogenesis of adult mice. The distribution of Hsp70-2 and Hsp70-2/lacZ mRNAs was determined by Northern blot, in situ hybridization, and RT-PCR, and it was found that upregulation of expression of both Hsp70-2 and Hsp70-2/lacZ was specific to the meiotic phase of spermatogenesis. (C) 1996 Academic Press, Inc.

gp130 is a ubiquitously expressed signal-transducing receptor component shared by interleukin 6, interleukin 11, leukemia inhibitory factor, oncostatin M, ciliary neurotrophic factor, and cardiotrophin 1. To investigate physiological roles of gp130 and to examine pathological consequences of a lack of gp130, mice deficient for gp130 have been prepared. Embryos homozygous for the gp130 mutation progressively die between 12.5 days postcoitum and term. On 16.5 days postcoitum and later, they show hypoplastic ventricular myocardium without septal and trabecular defect. The subcellular ultrastructures in gp130(-/-) cardiomyocytes appear normal. The mutant embryos have greatly reduced numbers of pluripotential and committed hematopoietic progenitors in the liver and differentiated lineages such as T cells in the thymus. Some gp130(-/-) embryos show anemia due to impaired development of erythroid lineage cells. These results indicate that gp130 plays a crucial role in myocardial development and hematopoiesis during embryogenesis.

The RBP-JK protein is a transcription factor that recognizes the sequence C(T)GTGGGGA, The RBP-J kappa gene is highly conserved in a wide variety of species and the Drosophila homologue has been shown to be identical to Suppressor of Hairless [Su(H)] which plays important roles in the development of the peripheral nervous system, To explore the function of the RBP-J kappa gene in mouse embryogenesis, a mutation was introduced into the functional RBP-J kappa gene in embryonic stem (ES) cells by homologous recombination, Null mutant ES cells survived but null mutant mice showed embryonic lethality before 10.5 days of gestation, The mutant mice showed severe growth retardation as early as 8.5 days of gestation, Developmental abnormalities, including incomplete turning of the body axis, microencephaly, abnormal placental development, anterior neuropore opening and defective somitogenesis, were observed in the mutant mice at 9.5 days of gestation, RBP-J kappa mutant embryos expressed a posterior mesodermal marker FGFR1. Their irregularly shaped somites expressed a somite marker gene Mox I but failed to express myogenin. The RBP-J kappa gene was revealed to be essential for postimplantation development of mice.

Background: Programmed cell death is an essential event during mammalian morphogenesis which eliminates unnecessary cells to accomplish histogenesis and organogenesis, Cell death in interdigital spaces of the developing limb is a classical example of morphogenetic cell death, We investigated whether classical programmed cell death in the interdigital tissue of the developing limb in mice is apoptosis with fragmentation of nuclear DNA and also examined sequentially the occurrence of programmed cell death and cell proliferation in the developing limb of mouse fetuses to analyze their interrelation. Methods: We examined the occurrence of apoptotic cell death in the developing limbs of mouse fetuses by using Nile blue sulphate staining, agarose gel electrophoresis for detecting DNA laddering, and a cytochemical labeling of DNA fragmentation. We also labeled proliferating cells using BrdU/anti-BrdU immunohistochemistry and examined the interrelation between apoptotic programmed cell death and cell proliferation. Results: DNA ladders, a biochemical evidence of apoptosis, were detected in DNA extracts from the interdigital tissue of day 13 mouse fetuses by agarose gel electrophoresis. Programmed cell death and DNA fragmentation were detected by Nile blue staining and cytochemical labeling of DNA fragmentation, respectively, in the interdigital mesoderm and in the regions of presumptive joints of the digit, BrdU/anti-BrdU immunohistochemistry for identifying proliferating S-phase cells revealed that interdigital mesenchymal cells cease DNA synthesis before programmed cell death and DNA fragmentation begin. Conclusions: We confirmed that both cytological apoptotic alterations and fragmentation of nuclear DNA occur in the interdigital tissue and presumptive joint areas of fetal mouse limbs, and they appear to play a significant role in the separation of digits as well as the formation of joint cavities. (C) 1995 Wiley-Liss, Inc.

HEPATOCYTE growth factor/scatter factor (HGF/SF) functions as a mitogen, motogen and morphogen for a variety of cultured cells(1-7). The genes for HGF/SF and its receptor (the c-met protooncogene product(8)) are expressed in many tissues during the embryonic periods and in the adult(9-14). HGF/SF is thought to mediate a signal exchange between the mesenchyme and epithelia during mouse development(15). To examine the physiological role of HGF/SF, we generated mutant mice with a targeted disruption of the HGF/SF gene. Here we report that homozygous mutant embryos have severely impaired placentas with markedly reduced numbers of labyrinthine trophoblast cells, and die before birth. The growth of trophoblast cells was stimulated by HGF/SF in vitro, and the HCF/SF activity was released by allantois in primary culture of normal but not mutant embryos. These findings suggest that HGF/SF is an essential mediator of allantoic mesenchyme-trophoblastic epithelia interaction required for placental organogenesis.

The fibrous sheath is a major cytoskeletal structure in the principal piece of the mammalian sperm flagellum. We have cloned a cDNA and used it to characterize the expression of mRNA for a mouse sperm fibrous sheath protein. Peptides from a tryptic digest of fibrous sheath proteins were separated by HPLC and a 31 amino acid sequence was obtained from one of the peptides. Through the use of degenerate oligonucleotide polymerase chain reaction (PCR) primers predicted from this sequence, an 80-bp product was amplified from mouse testis first-strand cDNA. This was utilized as a probe to isolate a 2.9-kb cDNA clone from a mouse round spermatid cDNA library. Sequence analysis of the cDNA clone showed that it encodes a protein with an open reading frame of 849 amino acids and includes the original peptide sequence. The predicted protein has a molecular weight of 93 795 and contains 32 cysteine residues and 32 potential phosphorylation sites. It has no significant homology with other known cytoskeletal proteins. Northern blot analysis detected an mRNA of similar to 3 kb that was abundant in round spermatids of the mouse and in testes from six other mammalian species, but not in twelve somatic tissues from the mouse. In situ hybridization analysis indicated that the mRNA is first detected in step I-G spermatids, is most abundant in step 8-12 spermatids, and decreases in amount in step 13-15 spermatids, suggesting that expression of the mRNA occurs in the postmeiotic phase of spermatogenesis. In addition, monoclonal antibody 5A8, which recognizes proteins of the mouse fibrous sheath, was used to isolate cDNAs from a mouse spermatid expression library that were found by PCR analysis to be homologous to the original cDNA clone. These results suggest that the similar to 3-kb mRNA encodes a major structural component of the mouse fibrous sheath that appears to be a unique cytoskeletal protein of spermatogenic cells. We designate the cDNA for this fibrous sheath component Fsc1.

The process of palate fusion was examined in 13- and 14-day-old mouse fetuses by using in situ staining for nuclear DNA fragmentation (TUNEL method) and immunofluorescent staining for keratin, with special reference to the disruption of the midline epithelial seam. TUNEL-positive cells were found in the disappearing midline seam and the oral and nasal epithelial triangles at some late stages of palate fusion, but not in the palatal shelves prior to contact or in the intact midline epithelial seam. It seems that DNA fragmentation or apoptosis is required for the midline epithelial seam to disrupt, but may not be necessary for initial contact of palatal shelves or for the epithelial fusion of opposing palatal shelves. A similar sign of apoptotic cell death was observed in the disappearing epithelial seam between the fusing nasal septum and dorsal palate. We have demonstrated that apoptotic programmed cell death does occur at some stages of palate fusion, although the present results do not exclude the possibility of epithelial-mesenchymal transformation and the oral and nasal migration of midline epithelial cells.

A fluorescent in situ hybridization (FISH) study was carried out on paraffin sections of human embryonic and fetal tissues with two DNA probes, DXZ1 and DYZ1 (Oncor), for X and Y chromosome-specific DNA sequences, respectively. The specificity of the DNA probes was confirmed on metaphase and interphase lymphocytes of healthy normal adult males. The paraffin blocks of the human embryonic and fetal tissues examined in the present study had been stored at room temperature for up to 5 years after fixation in 4% paraformaldehyde. All the seven embryos and five fetuses examined were successfully sexed by FISH. The cells from three embryos and four fetuses were positive for a hybridization signal with each of the DXZ1 and DYZ1 probes and they were classified as male. The cells from the remaining four embryos and one fetus were positive for two identical hybridization signals with the DXZ1 probe in a nucleus instead of the absence of the signal hybridized with DYZ1, indicating that their cells have two X chromosomes but no Y chromosomes. The FISH results for the five fetuses examined were consistent with their genital sex and/or gonadal histology. The FISH results were highly specific and no false positive or false negative results were obtained. Thus, the FISH technique has been shown to visualize specific DNAs in situ on paraffin sections and to be useful to determine the sex of fixed embryos and fetuses retrospectively. (C) 1994 Wiley-Liss, Inc.

We have identified cDNAs representing three hexokinase mRNAs (Hk1-sa, Hk1-sb, Hk1-sc) by screening mouse spermatogenic cell cDNA libraries with a mouse hepatoma cell line hexokinase (Hk1) cDNA [Arora KY, Fanciulli M, Pederson PL. J Biol Chem 1990; 265:6481-6488]. Although all three cDNAs show 99% identity to the somatic Hk1 cDNA sequence throughout most of their coding region, they differ from this sequence at the 5' end. They contain a common spermatogenic cell-specific sequence and a sequence unique to each cDNA immediately 5' to the common domain. However, they lack the porin-binding domain (PBD) present in this region of Hk1, used for binding to a pore-forming protein in the outer mitochondrial membrane. These observations appear to support a model proposed by others for hexokinase gene evolution in mammals. In addition, we found that Hk1-sb has an internal sequence that is not present in Hk1, Hk1-sa, or Hk1-sc. Moreover, Hk1-sa and Hk1-sb transcripts are developmentally expressed in mouse spermatogenic cells. Hk1-sa mRNA is first expressed during meiosis and continues to be present in postmeiotic germ cells, while the more abundant Hk1-sb mRNA is detected only in postmeiotic germ cells. These and other findings suggest that enzymes encoded by Hk1-sa, Hk1-sb, and Hk1-sc are present only in spermatogenic cells.

Two cases of double superior vena cava were found among 300 Japanese cadavers dissected at Kyoto University from 1980 to 1989. Case 1 was an 82-year-old male patient who died of cerebral infarction. Case 2 was a 39-year-old well-developed male patient who died of sepsis. They had no history of cardiovascular disorders. Common features in both cases are as follows: In addition to a right superior vena cava with normal appearance located in the normal position, on the left side, a normal-looking extra superior vena cava persisted and descended vertically in front of the aortic arch between the left pulmonary vein and the left auricle, traversed the coronary sulcus and finally drained into the right atrium. No differences in diameter were found between right and left venae cavae. No communicating vessels were present between them. Therefore, both cases, which had a persistent left superior vena cava draining into the coronary sinus, may be classified as McCotter's 1st type (1916) or Donadio's 3rd type (1925). Furthermore, in Case 2, the double azygos vein persisted symmetrically, suggesting the 4th type of Nandy and Blair (1965). In addition to venous anomalies, Case 2 had arterial anomalies such as the right subclavian artery arising as the last branch of the aortic arch similar to the G type of Adachi (1928), and the right vertebral artery arising from the right common carotid artery. The present cases are considered to be the 53rd and 54th cases of double superior vena cava found in Japanese cadavers, and the 18th and 19th cases of double superior vena cava without communicating vessels between them.

In our cadaver dissection, the connection between the lateral calcaneal artery, the lateral tarsal artery, and the branch of the lateral plantar artery was corroborated, indicating possible reverse blood flow in the lateral calcaneal artery. The distally based lateral calcaneal flap has been successfully used in 2 patients. Vascular anatomy, indication, and technical points of the surgery as well as advantages and disadvantages of the flap are discussed.