Hirokazu Fujimoto

The paternal methylation imprint of the mouse H19 locus is acquired in the gonocyte stage during foetal testis development

Germline‐specific differential DNA methylation that persists through fertilization and embryonic development is thought to be the ‘imprint’ distinguishing the parental alleles of imprinted genes. If such methylation is to work as the imprinting mechanism, however, it has to be reprogrammed following each passage through the germline. Previous studies on maternally methylated genes have shown that their methylation imprints are first erased in primordial germ cells (PGCs) and then re‐established during oocyte growth.

Cloning of a hsp70-related gene expressed in mouse spermatids

A cDNA library of spermatids was screened by a differential hybridization in order to isolate genes expressed in haploid cells of the mouse male germ line. A clone was found that encoded a protein related to the heat shock protein 70. A genomic DNA clone comparable to this cDNA clone was also isolated from a mouse genomic library. This gene had only one continuous open reading frame capable of coding a 630 amino-acid protein. There was an excellent match of the sequence with the heat shock protein 70 family but a difference from any previous 70 kilodalton heat shock protein. A 2.7kb transcript derived from this gene was expressed in spermatids but not in other testicular germ cells and somatic tissues. We have referred to this gene as hsc70t.

Effects of the Brachyury (T) mutation on morphogenetic movement in the mouse embryo

TT embryos have been first distinguishable at 8 days post coitum by their gross morphological abnormalities. By quantitative morphometry of histological sections, anomalies in the homozygotes were expressed numerically. At 8 days p.c., morphologically identifiable T-homozygotes had an increased number of ectodermal and a reduced number of mesodermal cells compared to the wild type. At 7 days p.c., embryos with a low mesoderm/ectoderm ratio were found only in litters of T+ × T+ matings at the expected frequency. At 6 days p.c., one-fourth of the embryos in T+ × T+ litters showed a delay in the transition from cuboidal to squamous endoderm. No such embryos were found in the +/+ × +/+ matings. In 6-, 7-, and 8-day mutant embryos, cells proliferated at statistically normal rates. Therefore, it may be said that advanced morphological irregularities of 8-day homozygotes cannot be accounted for by anomalies in cell proliferation. When the total cell number was 5 × 104/embryo (8 days), a sudden change was observed in the regional distribution of mesodermal and ectodermal cells along the anteroposterior axis of TT embryos. Since no regional difference in the cell cycle time was observed, these abnormalities may best be explained by anomalies in cell migration. These results strongly suggest abnormal morphology of TT mutants resulting from defects in morphogenetic movement.

Expression of human and mouse genes encoding polκ: testis-specific developmental regulation and AhR-dependent inducible transcription

Backgrounds Human polκ is a newly identified low‐fidelity DNA polymerase. While the enzyme bypasses an abasic site and acetylaminofluorene‐adduct in an error‐prone manner, it bypasses benzo[a]pyrene‐N2‐dG lesions in a mostly error‐free manner by incorporating predominantly dC opposite the bulky lesions. Benzo[a]pyrene (B[a]P) is activated through intracellular process mediated by the arylhydrocarbon receptor (AhR, also called the dioxin receptor), which is a ligand‐activated transcription factor with high affinities for aromatic compounds such as B[a]P and dioxin.

Expression of a mouse zinc finger protein gene in both spermatocytes and oocytes during meiosis

In order to identify genes regulating meiosis, a mouse spermatocyte cDNA library was screened for sequences encoding proteins with C2H2-type zinc finger motifs which are typically expressed by the Drosophila Krüppel gene. Three new cDNAs were isolated, and they were designated CTfin33, CTfin51, and CTfin92. Among them, CTfin51 was selected for further study. The deduced amino acid sequence revealed seven zinc finger motifs in its C-terminal region. Northern blot and in situ hybridization showed CTfin51 mRNA expression in spermatocytes after the pachytene stage and in early stage round spermatids of prepuberal and adult males. Immunocytochemical staining with an antiserum against beta-gal-CTfin51 fusion protein was localized within nuclei of spermatocytes and spermatids. Oocyte nuclei after the pachytene stage also were immunoreactive for CTfin51 protein. Immunoblots revealed a band at M(r) 75,000 in protein extracts from the testis and the ovary. These results suggest that the CTfin51 gene encodes a DNA-binding regulatory protein functionally associated with meiosis in both male and female gametogenesis.

The Hsp70 homolog gene, Hsc70t, is expressed under translational control during mouse spermiogenesis

Hsc70t is a member of the Hsp70 family of genes and is constitutively expressed after meiosis in mouse spermatogenesis. Immunohistochemistry and in situ hybridization techniques were used to examine the precise localization of the Hsc70t product during the various stages of spermatogenesis. A rabbit antiserum raised against the mouse Hsc70t‐lacZ fusion protein detected the Hsc70t protein in the late spermatid‐enriched fraction after two‐dimensional Western blot analyses. On histological sections, the protein appears in the cytoplasm of spermatids as they progress from step 9 to the final step of spermatogenesis. An antisense RNA probe generated from the 3′ untranslated region of Hsc70t cDNA detected Hsc70t mRNA in late round spermatids from step 7 onward with the signal disappearing in spermatids at step 15. Thus, Hsc70t mRNA first appears after meiosis in haploid cells but is not translated effectively until these cells progress to the transcriptionally inactive stage which coincides with chromatin condensation. These results establish that the synthesis of Hsc70t protein is under strict translational control. Mol. Reprod. Dev. 52:383–391, 1999.

Haploid specific activations of protamine 1 and hsc70t genes in mouse spermatogenesis

Protamine 1 and heat shock cognate 70 kDa protein (hsc70t) are known to be synthesized in haploid cells during spermatogenesis, and the mRNAs of these proteins have also been shown to accumulate in the haploid cells. However, it is unknown at which stage of spermatogenesis the genes for these proteins are actually activated. To examine this problem, we fractionated mouse adult testes cells at four different developmental stages, extracted their nuclei and carried out run-off assays with hsc70t and protamine 1 DNA probes. Results showed that both genes are mainly activated at the round spermatid stage. As the protein products of these genes accumulate at the later stage, it is interesting that these genes are regulated at the transcriptional and translational levels during spermatogenesis.

Inadequate function of sterile tw5/tw32 spermatozoa overcome by intracytoplasmic sperm injection

Mice carrying two t complementary haplotypes (tw5/tw32) are totally sterile. Their spermatozoa have poor motility and fertilize neither zone‐intact nor zona‐free oocytes, even though they are structurally indistinguishable from control (wild‐type) spermatozoa. However, when injected directly into oocytes, these infertile spermatozoa are able to participate in normal development. This suggests that infertility of tw5/tw32 male (spermatozoa) is more likely to be due to poor sperm‐oocyte interaction than to genetic incompetence of sperm nuclei.

The characterization of granular amoebocytes and their possible roles in the asexual reproduction of the polystyelid ascidian, Polyzoa vesiculiphora†‡

The blood cells in the bud and the zooid of the polystyelid ascidian, Polyzoa vesiculiphora, were examined by means of light and electron microscopy to identify the cells that have been named trophocytes. The large blood cells were abundant in the mesenchymal space of the bud, but not in that of the functional zooid. They contained glycogen particles, lipid droplets, large protein granules and autophagosomes in their cytoplasm and were identified as granular amoebocytes. The majority of these cells were specifically phagocytized by phagocytes during bud development and disappeared. These results indicate that the granular amoebocytes virtually represent trophocytes in Polyzoa and may participate in bud development via nutrient supply to the developing tissues.

Cloning of two isoforms of mouse DNA helicase Q1/RecQL cDNA; α form is expressed ubiquitously and β form specifically in the testis

Abstract We cloned cDNAs encoding mouse homologues for the human DNA helicase Q1/RecQL (human helicase Q1) which has homology with the Escherichia coli RecQ protein and found that they encode two isoforms. The two isoforms are identical over the entire sequence except for the carboxyl terminal sequence spanning less than 30 amino acids. One of the two isoforms, α, contains a sequence, KKRK, in the carboxyl terminus, which is also contained in human helicase Q1 and was confirmed to function as the nuclear localization signal. The other form, β, does not contain such a sequence. Expression of mouse helicase Q1 mRNA is extremely and relatively high in the testis and the thymus, respectively. RT-PCR analysis revealed that helicase Q1α was expressed in all tissues tested and the β form was expressed only in the testis. A survey of expression of Q1α and Q1β mRNA in the testis after birth revealed that Q1α mRNA is expressed in all testes of mice aged from 7 days to 8 weeks, and the expression of Q1β mRNA begins 14 days after birth, corresponding to the appearance of cells in the pachytene stage.