森 千里

The present study investigated the occurrence of apoptotic cell death in the mouse testis at various intervals following the administration of hydroxyurea (HU). The presence of apoptosis was assessed by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method and by DNA fragmentation assay using ligation-mediated polymerase chain reaction, Both the incidence of apoptotic cells and the level of DNA fragmentation in the testis increased depending on the HU dose, and they were most apparent at the highest dose (400 mg/kg). The incidence of apoptotic cells in the HU-treated group increased continuously and peaked at 12 h, but then decreased gradually, reaching control levels by 48 h. After HU treatment, TUNEL-positive apoptotic cells increased in the seminiferous epithelium of the tubules, and affected cells were found synchronously in the tubules of animals treated with HU. Spermatogonia and spermatocytes were found to be affected selectively. TUNEL-positive cells were found to be stage-specific and were primarily in stage IV-VI tubules. It has been shown that in vivo HU exposure induced testicular germ cell apoptosis dose dependently in a time- and stage-specific manner, and damaged cells appeared to be eliminated by phagocytosis by neighboring cells. Apoptosis of damaged testicular germ cells is apparently a common response to various testicular toxicants therefore protecting the next generations of germ cells from the damaged cell population. (C) 1999 Academic Press.

Tissue distribution and cellular localization of rhophilin, a 71 kDa Rho-binding protein, were examined in mice. Rhophilin mRNA was highly expressed in adult testis, but was absent in the testis of WIWV mice deficient in germ cells.,An anti-rhophilin antibody detected a band of an expected size in sperm extracts, which was enriched in the tail fraction. Immunofluorescence analysis revealed two lines of striated staining running in parallel in the principal piece of the sperm tail, These results suggest that rhophilin is expressed in germ cells and localized in the fibrous sheath of the sperm tail. (C) 1999 Federation of European Biochemical Societies.

Cisplatin and VP-16 were used to study the induction of apoptosis in Panc-1 cells. Cisplatin and VP-16 inhibited the growth of Panc-1 cells. After 2 hours exposure to cisplatin or VP-16, attached and detached cells were subjected to TUNEL staining to calculate the ratio of apoptosis. In detached cells TUNEL positive ratios increased in a time-and dose-dependent manner In Western blotting, Bm expression was obviously upregulated but Bcl-2 remained almost constant. The results suggested that in Panc-1 cells cisplatin and VP-16 induced apoptotic cell death which was mediated through the interaction of Bax expression in the presence of mutated p53.

It has been recognized in mammals that after pancreatic duct obstruction, acinar cells progressively disappear and pancreatic islets are preserved. Previous studies by electron microscopy have suggested the involvement of apoptosis in acinar cell deletion. In the present study, we employed molecular biological methods and investigated whether acinar cell deletion is due to apoptosis. In male Sprague-Dawley rats, pancreatic duct Ligation was performed through a left paramedian incision. Pancreatic tissue was studied at each of the following intervals after ligation: 3, 6, 12, 18, and 24 h and 2, 3, 5, and 7 days. DNA fragmentation was determined by in situ labeling of DNA strand breaks on tissue sections [fluorescein-labeled terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling method (TUNEL)] and by electrophoretic detection of the fragments of extracted DNA. Tissue sections were also examined by hematoxylin/eosin staining and immunohistochemical staining of insulin. Pancreatic duct ligation induced acinar cell deletion by day 5. Pancreatic tissue from control rats demonstrated no TUNEL-positive nuclei. In contrast, acinar cells from rats 12 h to 5 days after duct ligation showed TUNEL-positive nuclei. The number of TUNEL-positive nuclei was maximum 2 days after duct ligation. Electrophoresis showed DNA fragmentation after duct ligation. Control rats showed a genomic DNA pattern. Islets were preserved throughout the experimental period in duct-ligated rats and control rats. The results suggest that apoptosis may be the dominant form of acinar cell death in the rat pancreatic duct ligation model.

The present study examined the occurrence of apoptotic cell death in the testis of wild-type mice from postnatal days 3 to 28 and in juvenile Hsp70-2 knockout mice. Adult Hsp70-2 knockout males are infertile and lack spermatids and spermatozoa (Dir et al. [1996a] Proc. Natl. Acad. Sci. U.S.A. 93:3264-3268). To identify the cell types undergoing apoptosis, we also examined the relationship between the occurrence of apoptotic cell death and the expression pattern of the Hsp70-2 gene product (heat-shock protein 70-2 [Hsp70-2]; marker for spermatocytes and spermatids), germ cell nuclear antigen 1 (GCNA1; marker for spermatogonia and spermatocytes), and vimentin (marker for Sertoli cells). This study shows that during postnatal development of the wildtype mouse testis (1) the percentage of apoptotic cell death detected by the TdT-mediated dUTP-biotin nick end labeling (TUNEL) method is higher in mice from days 8 to 22 than in younger or older mice, (2) the majority of apoptotic cells are spermatogonia and less frequently are spermatocytes, and (3) the degenerative cell death of spermatogonia and primary spermatocytes involves apoptosis with fragmentation of DNA. The analysis of apoptotic cell death in the testes of juvenile Hsp70-2 knockout mice showed an additional increased level of apoptosis at day 17, during the first wave of spermatogenesis, in pachytene spermatocytes. (C) 1997 Wiley-Liss, Inc.

Several enzymes in the glycolytic pathway are reported to have spermatogenic cell-specific isozymes. We reported recently the cloning of cDNAs representing three unique type 1 hexokinase mRNAs (mHk1-sa, mHk1-sb, and mHk1-sc) present only in mouse spermatogenic cells and the patterns of expression of these mRNAs (Mori et al., 1993: Biol Reprod 49:191-203). The mRNAs contain a spermatogenic cell-specific sequence, but lack the sequence for the porin-binding domain that somatic cell hexokinases use to bind to a pore-forming protein in the outer mitochondrial membrane. We now report the cloning of cDNAs representing three unique human type 1 hexokinase mRNAs (hHK1-ta, hHK1-tb, and hHK1-tc) expressed in testis, but not detected by Northern analysis in other human tissues. These mRNAs also contain a testis-specific sequence not present in somatic cell type 1 hexokinase, but lack the sequence for the porin-binding domain. The hHK1-tb and hHK1-tc mRNAs each contain an additional unique sequence. The testis-specific sequence of the human mRNAs is similar to the spermatogenic cell-specific sequence of the mouse mRNAs. Furthermore, Northern analysis of RNA from mouse, hamster, guinea pig, rabbit, ram, human, and rat demonstrated expression of type 1 hexokinase mRNAs lacking the porin-binding domain in the testes of these mammals. These results suggest that hexokinase may have unique structural or functional features in spermatogenic cells and support a model proposed by others for hexokinase gene evolution in mammals. (C) 1996 Wiley-Liss, Inc.

In addition to the five 70-kDa heat shock proteins (HSP70) common to germ cells and somatic tissues of mammals, spermatogenic cells synthesize HSP70-2 during meiosis, To determine if this unique stress protein has a critical role in meiosis, we used gene-targeting techniques to disrupt Hsp70-2 in mice. Male mice homozygous for the mutant allele (Hsp70-2(-/-)) did not synthesize HSP70-2, lacked postmeiotic spermatids and mature sperm, and were infertile, However, neither meiosis nor fertility was affected in female Hsp70-2(-/-) mice, We previously found that HSP70-2 is associated with synaptonemal complexes in the nucleus of meiotic spermatocytes from mice and hamsters. While synaptonemal complexes assembled in Hsp70-2(-/-) spermatocytes, structural abnormalities became apparent in these cells by late prophase, and development rarely progressed to the meiotic divisions, Furthermore, analysis of nuclei and genomic DNA indicated that the failure of meiosis in Hsp70-2(-/-) mice was coincident with a dramatic increase in spermatocyte apoptosis. These results suggest that HSP70-2 participates in synaptonemal complex function during meiosis in male germ cells and is linked to mechanisms that inhibit apoptosis.

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 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.