Senkiti Sakai

A Chemokine, Interferon (IFN)-γ-Inducible Protein 10 kDa, Is Stimulated by IFN-τ and Recruits Immune Cells in the Ovine Endometrium

Abstract Proper distribution of immune cells in the uterus is a prerequisite for successful implantation and subsequent placentation, but biochemical signals that govern such events have not been well characterized. In the present study, the cDNA of a chemokine, interferon (IFN)-γ-inducible protein 10 kDa (IP-10), was identified from a cDNA subtraction study between uterine endometrial tissues from Day 17 pregnant and Day 15 cyclic ewes. The effect of IFN-τ on IP-10 expression and the involvement of IP-10 in the recruitment of immune cells were then investigated. Northern blot analysis revealed that large amounts of IP-10 mRNA were present during conceptus attachment to maternal endometrium and early placentation. IP-10 mRNA was localized to monocytes distributed in the subepithelial stroma of pregnant but not cyclic uteri. This finding was supported by the discovery of IP-10 mRNA expression in monocytes but not in lymphocytes, uterine epithelial cells, or stromal cells. Moreover, the expression of IP-10 mRNA by the monocytes was stimulated by IFN-α, IFN-γ, and IFN-τ in a dose-dependent manner, but the expression of IP-10 mRNA by the endometrial explants was most stimulated by IFN-τ. In a chemotaxis assay, migration of peripheral blood mononuclear cells was stimulated by the addition of IFN-τ stimulated-endometrial culture medium, and the effect was significantly reduced by neutralization with an anti-IP-10 antibody. These results suggest that endometrial IP-10 regulated by conceptus IFN-τ regulates recruitment and/or distribution of immune cells seen in the early pregnant uterus.

Posttranscriptional Regulation of Cyclin A1 and Cyclin A2 During Mouse Oocyte Meiotic Maturation and Preimplantation Development

Abstract A shift from a meiotic cell cycle to a mitotic cell cycle occurs following fertilization. The molecular basis for this transition, however, is poorly understood. Although cyclin A1 is proposed to regulate M phase in the meiotic cell cycle, and cyclin A2 is proposed to regulate S and M phases in the mitotic cell cycle, little is known about changes in the expression levels of cyclin A1 and A2 during meiotic and mitotic cell cycles in mammalian oocytes. We report that the mRNA levels of both cyclins A1 and A2 decrease during oocyte maturation. The amount of cyclin A1 mRNA then increases between the one-cell and blastocyst stages, whereas that of cyclin A2 remains relatively constant. The amount of cyclin A1 protein declines during maturation and is not readily detected from the two-cell to the blastocyst stage. In contrast, cyclin A2 is not readily detected in the oocyte and metaphase II-arrested egg but is detected following fertilization and throughout the subsequent stages of preimplantation development. The appearance of cyclin A2 protein following fertilization positively correlates with an increase in the size of the mRNA. This increase, as well as the increase in the amount of cyclin A2 protein, is prevented by 3′-deoxyadenosine (3′-dA), an inhibitor of polyadenylation. Consistent with a role for cyclin A2 in regulating the G1/S transition, 3′-dA also inhibits DNA replication in treated one-cell embryos. These results suggest that regulation of expression of cyclins A1 and A2 is under posttranscriptional regulation and that the observed changes in their expression may be involved in the transformation of a meiotic cell cycle to a mitotic cell cycle following fertilization.

The poly(A) tail length of casein mRNA in the lactating mammary gland changes depending upon the accumulation and removal of milk.

The length of casein mRNA from the lactating mouse mammary gland, as assessed on Northern blots, is shorter after weaning, but is elongated following the removal of milk. In order to investigate this phenomenon, the molecular structures of beta- and gamma-casein mRNAs were analysed. The coding and non-coding regions of the two forms were the same length, but the long form of casein mRNA had a longer poly(A) tail than the short form (P<0.05). In order to examine the stability of casein mRNA under identical conditions, casein mRNAs with the long and short poly(A) tails were incubated in the rabbit reticulocyte lysate (RRL) cell-free translation system. Casein mRNA with the long poly(A) tail had a longer half-life than that with the short tail (P<0.05). The beta- and gamma-casein mRNAs were first degraded into 0.92 and 0.81 kb fragments respectively. With undegraded mRNA, the poly(A) tail shortening by exoribonuclease was not observed until the end of the incubation. Northern blot analysis showed that casein mRNA with the long poly(A) tail was protected efficiently from endoribonucleases. We conclude that the length of the poly(A) tail of casein mRNA in the lactating mammary gland changes depending upon the accumulation and removal of the glands milk, and we show that the longer poly(A) tail potentially protects the mRNA from degradation by endoribonucleases.

l-Amino acid oxidase plays a crucial role in host defense in the mammary glands

The innate immune system plays an important role in protecting organs that are continuous with the outer surface of the body from bacterial infection. The antibacterial factors involved in this system have been sought in exocrine glands, particularly in the mammary glands. Because milk produced in the mammary glands is enriched in various nutrients, supporting the proliferation of bacteria, mammary glands appear to be at the greatest risk of bacterial infection and proliferation. Here, we show that mouse milk contains L‐amino acid oxidase (LAO), a lactating mammary gland‐specific protein that displays antibacterial activity in vitro through the production of hydrogen peroxide from free amino acids. We produced LAO‐disrupted mouse lines to define the physiological properties and importance of the protein in vivo. The LAO‐knockout mice were healthy and had normal mammary gland development;however, the antibacterial activity normally observed in milk from wild‐type mice was absent from the milk of knockout mice. The content of free amino acids targeted by LAO was very low in wild‐type milk, whereas these amino acids were abundant in LAO‐knockout milk. Knockout mice exhibited weak resistance to an intramammary bacterial challenge compared to their wild‐type counterparts. Further, preadministration of wild‐type milk whey reduced the severity of bacterial infection in LAO‐knockout mice. These results demonstrate that milk LAO protects the mammary gland against bacterial infection, and this antibacterial effect may be due to the generation of hydrogen peroxide by using free amino acids abundantly present in milk. — Nagaoka, K.,Aoki, F., Hayashi, M., Muroi, Y., Sakurai, T., Itoh, K., Ikawa, M., Okabe, M., Imakawa, K., Sakai, S. L Amino acid oxidase plays a crucial role in host defense in the mammary glands. FASEBJ. 23, 2514–2520 (2009)

Regulation of flagellar bending by cAMP and Ca2+ in hamster sperm

Hamster sperm were immotile in the medium at free Ca2+ concentrations ([Ca2+]) below 1 × 10–4 M. The flagellum was acutely bent in the opposite direction to the curve of the hook‐shaped heads. This phenomenon seemed to be caused by the decrease in the intracellular cAMP concentration, since the cAMP concentration was low at [Ca2+] below 1 × 10–4 M and increased abruptly at 1 × 10–3 M, at which sperm were swimming actively. In addition, sperm became motile due to treatment with 8‐bromo‐cAMP, a membrane permeable analogue of cAMP, in a medium without Ca2+. These results suggested that extracellular Ca2+ is involved in the regulation of flagellar movement via increasing intracellular cAMP concentration. By the treatment with W‐13, a calmodulin inhibitor, sperm also became motile, although cAMP concentration remained at a low level. These results suggested that cAMP is not always required for the flagellar movement when the function of calmodulin is depressed. Mol. Reprod. Dev. 53:77–83, 1999.

Localization of Janus Kinase 2 to the Nuclei of Mature Oocytes and Early Cleavage Stage Mouse Embryos

Abstract Jak2, which is a member of the Janus tyrosine kinase family, plays essential roles in cytokine signal transduction and in the regulation of cell growth and gene expression. To investigate the involvement of Jak2 in the regulation of early preimplantation development, we examined the expression of Jak2 in mouse embryos. Reverse transcription-polymerase chain reaction assays revealed that the relative amount of Jak2 mRNA was highest in unfertilized oocytes, gradually decreased until the four-cell stage, and remained at low levels until the blastocyst stage. Immunocytochemistry showed that Jak2 was localized predominantly to the female pronucleus in one-cell embryos. The immunofluorescence signal was very weak or undetectable in the male pronucleus. In unfertilized oocytes and one-cell embryos at M phase, Jak2 was localized to the chromosomes. After cleavage to the two-cell stage, the intensity of the immunofluorescence signal decreased in the nucleus while the embryos were in late G2. This decrease was independent of DNA synthesis because it was not affected by inhibition of DNA replication. However, inhibition of protein synthesis repressed the disappearance of Jak2 from the nucleus. These results suggest a novel function for Jak2 in the regulation of early preimplantation development.

Generation of hydrogen peroxide by a low molecular weight compound in whey of Holstein dairy cows.

Using an ultrafiltration membrane (molecular cut-off, 3,000), low molecular weight compounds in bovine milk were collected (YM-3 filtrate). A hydrogen peroxide (H2O2)-like substance was generated in the YM-3 filtrate. This substance was undetected at 0 h, but increased in a time-dependent manner, peaking after 2 h of incubation at 38 degrees C. After incubating the YM-3 filtrate with catalase and lactoperoxidase, the signal showing the presence of this substance disappeared. The substance was quantified using one chemiluminescence and three colorimetric H2O2 detection systems. In all systems, their estimates were within the same range. The amount of substance, as estimated by the chemiluminescence H2O2 detection system, was correlated with that estimated by the other three colorimetric systems (r=0.98, 0.95 and 0.87). The substance was eluted at the same position as H2O2 by gel filtration on Superdex 30. Thus, the substance had the same characteristics as H2O2. An H2O2-generating substance in either the YM-3 filtrate or whey had a molecular mass of about 600. In this study, we clarify that bovine milk is capable of generating H2O2 by utilizing a low molecular weight compound. Thus, we present a new type of H2O2-supplying system in bovine milk.

Involvement of the STAT5 Signaling Pathway in the Regulation of Mouse Preimplantation Development

Abstract The signal transducer and activator of transcription 5 (STAT5) is an essential factor in the signal transduction pathways for a number of cytokines that regulate the growth and differentiation of mammalian cells. In this study, we investigated the STAT5 signaling pathway in mouse embryos, to elucidate the mechanism of cytokine signal transduction that regulates preimplantation development. The results of the RT-PCR analysis showed that both STAT5A and B were expressed throughout preimplantation development. Immunocytochemistry revealed that the STAT5A/B proteins were located in the nucleus from the early 1-cell stage to the blastocyst stage. STAT5 activation appeared to be regulated by Janus kinases (JAKs) and SRC family kinases (SFKs), since inhibitors of these kinases inhibited the localization of STAT5 proteins to the nucleus. The JAK inhibitor Ag490 reduced both the developmental rate of the embryos and the expression levels of the downstream genes of the JAK-STAT5 signaling pathway. These findings suggest that STAT5 proteins function in preimplantation development by mediating the signals from cytokines.

The casein mRNA decay changes in parallel with the poly(A) tail length in the mouse mammary gland

Using beta- and gamma-casein mRNAs, the relationship between poly(A) tail length and half-life of mRNA is determined in the mouse mammary gland during pregnancy and lactation. beta- and gamma-Casein mRNAs increase before and after parturition, respectively. The poly(A) tail as well as the half-life of casein mRNA becomes longer upon the active casein mRNA synthesis. The poly(A) tail is shortened gradually as lactation progresses. The half-life of mRNA decreases approximately from 20 h at early to 4 h at late lactation. Northern blot analysis reveals that nuclear RNA has the same poly(A) tail length as casein mRNA in the cytoplasm does. Thus, the mammary gland changes the poly(A) tail length of casein mRNA. The poly(A) tail length changes in parallel with the level of poly(A) polymerase (PAP) mRNA during pregnancy and lactation, suggesting that the mammary gland determines the poly(A) tail length of casein mRNA through the change in the PAP gene expression. As the half-life of casein mRNA is related with the degree of polyadenylation, we conclude that the poly(A) tail elongation and shortening is a mechanism in regulating the mRNA decay.

Binding of prolactin and monoclonal antibody to prolactin receptors immobilized on a nitrocellulose membrane filter

Mammary prolaction (PRL) receptors in 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonic acid (Chaps) extract were immobilized on a nitrocellulose (NC) filter, and a binding assay using the filter-bound receptors was carried out in the absence of detergent. PRL binding to the receptors was dependent on the quantity of the receptors and the PRL added to the filters. The filter-bound receptors retained the specificity of binding to peptide hormones. Scatchard analysis showed that the number of PRL receptors and the dissociation constant for PRL binding are essentially unchanged after immobilization on a NC filter, indicating that the PRL binding site does not participate in the binding to the NC filter and is equally available for PRL binding. The monoclonal antibody (MAb) against the PRL receptor was able to bind specifically to the Chaps-solubilized and filter-bound PRL receptors, as shown by curvilinear Scatchard plots. Immobilization on NC filters permits direct detection and characterization of the soluble PRL receptor using labeled PRL or MAb.