Yoshikage Muroi

Induction of Endogenous Interferon Tau Gene Transcription by CDX2 and High Acetylation in Bovine Nontrophoblast Cells

Abstract Interferon tau gene (IFNT) is expressed only by mononuclear trophectoderm cells in ruminant ungulates. To our knowledge, its epigenetic regulation and interaction with trophectoderm lineage-specific caudal-related homeobox 2 transcription factor (CDX2) have not been characterized. Herein, we studied differences in chromatin structures and transcription of endogenous bovine IFNT in bovine trophoblast BT-1 and CT-1 cells and in nontrophoblast MDBK cells. Transcripts from endogenous IFNT and CDX2 genes were found in BT-1 and CT-1 cells but not in MDBK cells. Chromatin immunoprecipitation study revealed that CDX2 binding sites exist in proximal upstream regions of IFNT (IFN-tau-c1). Endogenous IFNT transcription in BT-1 cells was increased with CDX2 overexpression but was reduced with short interfering RNA specific for the CDX2 transcript. In chromatin immunoprecipitation studies, histone H3K18 acetylation of IFNT was higher in CT-1 cells than in MDBK cells, while histone H3K9 methylation was lower in CT-1 cells than in nontrophoblast cells. In MDBK cells (but not in CT-1 cells), histone deacetylases were bound to IFNT, which was reversed with trichostatin A treatment; treatment with trichostatin A and CDX2 then increased IFNT mRNA levels that resulted from abundant CDX2 mRNA expression. These data provide evidence that significant increase in endogenous IFNT transcription in MDBK cells (which do not normally express IFNT) can be induced through CDX2 overexpression and high H3K18 acetylation, but lowering of H3K9 methylation could also be required for the degree of IFNT transcription seen in trophoblast cells.

Involvement of GATA transcription factors in the regulation of endogenous bovine interferon‐Tau gene transcription

Expression of interferon‐tau (IFNT), necessary for pregnancy establishment in ruminant ungulates, is regulated in a temporal and spatial manner. However, molecular mechanisms by which IFNT gene transcription is regulated in this manner have not been firmly established. In this study, DNA microarray/RT‐PCR analysis between bovine trophoblast CT‐1 and Mardin–Darby bovine kidney (MDBK) cells was initially performed, finding that transcription factors GATA2, GATA3, and GATA6 mRNAs were specific to CT‐1 cells. These mRNAs were also found in Days 17, 20, and 22 (Day 0 = day of estrus) bovine conceptuses. In examining other bovine cell lines, ovary cumulus granulosa (oCG) and ear fibroblast (EF) cells, GATA2 and GATA3, but not GATA6, were found specific to the bovine trophoblast cells. In transient transfection analyses using the upstream region (−631 to +59 bp) of bovine IFNT gene (bIFNT, IFN‐tau‐c1), over‐expression of GATA2/GATA3 did not affect the transcription of bIFNT‐reporter construct in human choriocarcinoma JEG3 cells. Transfection of GATA2, GATA3, ETS2, and/or CDX2, however, was effective in the up‐regulation of the bIFNT construct transfected into bovine oCG and EF cells. One Point mutation studies revealed that among six potential GATA binding sites located on the upstream region of the bIFNT gene, the one next to ETS2 site exhibited reduced luciferase activity. In CT‐1 cells, endogenous bIFNT gene transcription was up‐regulated by over‐expression of GATA2 or GATA3, but down‐regulated by siRNA specific to GATA2 mRNA. These data suggest that GATA2/3 is involved in trophoblast‐specific regulation of bIFNT gene transcription. Mol. Reprod. Dev. 76: 1143–1152, 2009.

Identification of Novel Endogenous Betaretroviruses Which Are Transcribed in the Bovine Placenta

ABSTRACT Sequences of retroviral origin occupy approximately 10% of mammalian genomes. Various infectious endogenous retroviruses (ERVs) and functional retroviral elements have been reported for several mammals but not cattle. Here, we identified two proviruses, designated bovine endogenous retrovirus K1 (BERV-K1) and BERV-K2, containing full-length envelope (env) genes in the bovine genome. Phylogenetic analysis revealed that they belong to the genus Betaretrovirus. By reverse transcription (RT)-PCR, both BERV-K1 and -K2 env mRNAs were detected in the placenta and cultured bovine trophoblast cells. Real-time RT-PCR analysis using RNAs isolated from various bovine tissues revealed that BERV-K1 env mRNA was preferentially expressed in the placenta. Moreover, we also found the expression of doubly spliced transcripts, named the REBK1 and REBK2 genes. Both the REBK1 and REBK2 proteins have motifs for a putative nuclear localization signal and a nuclear export signal. REBK1 and REBK2 fused with green fluorescent proteins were localized mainly in the nuclei when they were expressed in bovine and porcine cells. In the env and 3′ long terminal repeats of BERV-K1 and -K2, we found regulatory elements responsible for the splicing and transport of viral RNAs and/or translation of the env genes. Although we have not identified the expressed Env proteins in bovine tissues, these data suggest that both BERV-K1 and BERV-K2 express Env proteins and that these proteins may have physiological functions in vivo.

Volatile female odors activate the accessory olfactory system of male mice without physical contact

We previously reported that male mice are more attracted to volatile odors from intact female mice than from ovariectomized female mice. In the present study, we investigated male attraction to volatile odors from soiled bedding collected from the cages of estrous or ovariectomized female mice. There was no difference in the total time spent sniffing volatile odors from estrous and ovariectomized female mice, suggesting that female mice emit volatile odors which are not excreted into bedding. To test this possibility, we investigated c-Fos expression in the mitral cell layer and granule cell layer of the accessory olfactory bulb 60 min after exposure of male mice to volatile odors without physical contact. Volatile odors from an estrous female mouse significantly increased the total number of c-Fos positive cells in each of the rostral and caudal granule cell layer, but not in the mitral cell layer. After exposure to volatile odors from estrous bedding, the total number of c-Fos positive cells did not increase. Volatile odors from a male mouse did not increase the total number of c-Fos positive cells. Volatile odors from an ovariectomized female mouse increased c-Fos expression only in the caudal granule cell layer. These results suggest that female mice emit specific volatile odors which are not excreted into bedding, and that the volatile odors activate the accessory olfactory system of male mice without physical contact. To characterize the female-specific volatile odors, we conducted habituation-dishabituation tests. Whereas sham-operated male mice discriminated between volatile odors of estrous and ovariectomized female mice, vomeronasal organ-removed male mice did not. These results suggest that male mice discriminated whether or not female mice were ovariectomized, by volatile odors via the accessory olfactory system, and that the female-specific volatile odors are involved in reproduction.

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)

CD9 regulates transcription factor GCM1 and ERVWE1 expression through the cAMP/protein kinase A signaling pathway

ERVWE1 (SYNCYTIN-1), a membrane protein originating from the envelope gene of human endogenous retrovirus-W (HERV-W), mediates the fusion of mononucleated cytotrophoblasts into multinucleated syncytiotrophoblast. Though ERVWE1 has been characterized since its discovery, regulatory mechanisms associated with ERVWE1 expression have not been firmly established. We hypothesized that membrane protein CD9, involved in cell-cell fusion of fertilization and myogenesis, could be involved in the regulation of ERVWE1 gene expression. In this study, regulatory mechanisms of ERVWE1 expression were studied using human choriocarcinoma BeWo cells. Forskolin is an activator of adenylate cyclase, which increased CD9 and ERVWE1 expression. The increase in CD9 expression was inhibited by a protein kinase A (PKA) inhibitor, Rp-cAMPS. These results indicate that CD9 expression is regulated by the cAMP/PKA signaling pathway. Overexpression of CD9 increased expression levels of ERVWE1 as well as GCM1 (hGCMa), which is a transcription factor known to activate ERVWE1 gene transcription. However, high ERVWE1 expression induced by CD9 overexpression did not result in the increase in chorionic gonadotropin, beta polypeptide production. Moreover, CD9-induced increase in ERVWE1 and GCM1 expressions were inhibited by Rp-cAMPS. These results suggest that CD9 increases GCM1 expression via the cAMP/PKA signaling pathway, resulting in the increase in ERVWE1 expression.

Oligomannose-coated liposome-entrapped dense granule protein 7 induces protective immune response to Neospora caninum in cattle

Neospora caninum is an intracellular protozoan parasite that causes abortion in cows. Vaccination is an important strategy for control of neosporosis, and a safe and effective vaccine suitable for cattle is required. Dense granule protein 7 of N. caninum (NcGRA7) is a secretory protein with high antigenicity in hosts. We demonstrated previously that NcGRA7 entrapped in liposomes coated with mannotriose (M3-NcGRA7) could induce a parasite-specific T-helper type 1 immune response and produce humoral antibodies that resulted in increased offspring survival and decreased infection in the brains of mice dams. In the present study, the efficacy of M3-NcGRA7 as a vaccine candidate against N. caninum has been evaluated in cattle (n=12). Cattle were immunized with M3-NcGRA7 containing 50 μg (n=4) or 200 μg NcGRA7 (n=4) subcutaneously twice with a 4-week interval and all cattle including the non-immunized controls (n=4) were inoculated with 10(7) tachyzoites of Nc-1 strain 27 days after the second immunization and euthanized at 85-87 days post infection (dpi). In immunized cattle, NcGRA7-specific antibody production and IFN-γ production in PBMC was induced before challenge. At 3 dpi, body temperature and concentration of serum IFN-γ tended to be higher in control cattle than in the immunized cattle. Furthermore, the parasite load in the brain significantly decreased in cattle immunized with 50 μg M3-NcGRA7 compared with controls. These results suggest that M3-NcGRA7 can induce protective immune responses to N. caninum tachyzoites in cattle, which could lead to practical application of safe and effective subunit vaccines.

A competitive effect of androgen signaling on male mouse attraction to volatile female mouse odors.

Olfaction plays an important role in animal communication. We hypothesized that males recognize the attractive volatile odors attributed to female reproductive ability. We measured the period during which a male mouse spent sniffing volatile odors from a sham-operated female mouse or an ovariectomized mouse without visual or tactile contact. Intact male mice spent more time sniffing volatile odors from proestrous, estrous or metestrous females than from ovariectomized females. There was no difference in castrated male mice. To investigate the involvement of sexual hormone in this behavior, castrated male mice were treated with 17 alpha-estradiol (E), dihydrotestosterone (DHT), or both. E-treatment did not affect sniffing behavior. Regardless of the estrous stages, DHT-treated castrated males spent less time sniffing the volatile odors from sham-operated than from ovariectomized female mice. Both E- and DHT-treated castrated males spent less time sniffing the volatile odors from proestrous or estrous females than from ovariectomized females. These results suggest that neither androgen nor estrogen is sufficient for reproducing male attraction to volatile female mouse odors, and that androgen signaling has a competitive effect against the attraction.

Tissue Distribution of Neospora caninum in Experimentally Infected Cattle

ABSTRACT Histopathology and quantitative PCR (qPCR) were used to determine the tissue distribution of Neospora caninum in calves at 80 days postinfection. Our findings revealed that the most appropriate brain areas for researching N. caninum pathogenesis were the amygdala and hippocampus for qPCR and the corpus striatum and diencephalon for histopathology.

A novel neuropeptide Y neuronal pathway linking energy state and reproductive behavior

Animals consume energy for reproduction, as well as survival. Excess or insufficient energy investment into reproduction, respectively, threatens the survival of parents or leads to the failure of reproduction. Management of energy consumption in reproduction is important, not only for the success of the process, but also for the survival of the parents. Reproductive behaviors, such as mating and parental behavior, are indispensable for achieving each event of reproduction including gametogamy, parturition, and lactation. Therefore, reproductive behavior is one of the important factors in managing energy consumption for reproduction. Orexigenic and anorexigenic molecules in the hypothalamus have been implicated in the regulation of reproductive functions. An orexigenic neuropeptide, neuropeptide Y (NPY), has been also implicated in the regulation of both reproduction and energy state of animals. In this review, we will first summarize the neuronal mechanism for regulating reproductive functions by orexigenic and anorexigenic molecules in the hypothalamus. Second, we will focus on the NPY neuronal pathways regulating reproductive behavior in the intra- and extra-hypothalamic brain areas. We will highlight the NPY neuronal pathway from the arcuate nucleus to the dorsal raphe nucleus as a novel extra-hypothalamic pathway for energy state-dependent regulation of reproductive behavior. Finally, we will propose a biological significance of the extra-hypothalamic NPY neuronal pathway, which plays an important role in the associative control of feeding and reproductive behaviors.