No abstract

For a pregnancy to be established, initial apposition and adhesion of the blastocyst to maternal endometrium must occur in a coordinated manner; however, a key factor(s) that mediates the trophoblast cell migration and attachment to the apical surface of the endometrium has not been identified. In this study, we examined the effect of an endometrial chemokine, interferon-␥-inducible protein 10 kDa (IP-10), on conceptus migration to the endometrial epithelium. We first studied endometrial IP-10 mRNA expression, which was localized in the subepithelial stromal region, and detected the protein in the uterine flushing media during early pregnancy. Expression of IP-10 mRNA by the endometrium of cyclic animals was stimulated by the addition of a conceptus factor interferon-tau (IFN-). Immunofluorescent analysis revealed that IP-10 receptor, CXCR3, was localized in the trophoblast cells, to which biotinylated-recombinant caprine IP-10 (rcIP-10) bound. Chemotaxis assay indicated that rcIP-10 stimulated the migration of trophoblast cells, and the effects of rcIP-10 were neutralized by the pretreatment with an anti-IP-10 antibody. Adhesive activity of trophoblast cells to fibronectin was promoted by rcIP-10, and the effect was inhibited by the use of anti-IP-10 antibody. Further adhesion experiments demonstrated that binding of trophoblast cells to fibronectin was completely inhibited by a peptide of the Arg-Gly-Asp (RGD) sequence, which binds to integrins ␣ 5 ␤ 1 , ␣ V ␤ 1 , ␣ V ␤ 3 , and ␣ V ␤ 5 , whereas non-binding peptide containing Arg-Gly-Glu (RGE) had minimal effects. More importantly, rcIP-10 promoted the adhesion of trophoblast cells to primary cells isolated from endometrial epithelium. Furthermore, rcIP-10 stimulated the expression of integrin ␣ 5 , ␣ V , and ␤ 3 subunit mRNA in trophoblast cells. These findings suggest that endometrial IP-10 regulates the establishment of apical interactions between trophoblast and epithelial cells during early gestation.

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)-gamma-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-tau 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-alpha, IFN-gamma, and IFN-tau in a dose-dependent manner, but the expression of IP-10 mRNA by the endometrial explants was most stimulated by IFN-tau. In a chemotaxis assay, migration of peripheral blood mononuclear cells was stimulated by the addition of IFN-tau 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-tau regulates recruitment and/or distribution of immune cells seen in the early pregnant uterus.

L-Amino acid oxidase (LAO) was purified from mouse milk. LAO reacted with L-amino acids in an apparent order of Phe > Met, Tyr > Cys, Leu > His > > other 11 amino acids tested and produced H 2 O 2 in a dose-and time-dependent manner. LAO in milk had a molecular mass of about 113 kDa and was converted to a 60-kDa protein by SDS-PAGE. LAO consisted of two subunits. The N-and C-terminal amino acid sequence determination followed by cDNA cloning showed that the 60-kDa protein consisted of 497 amino acids. LAO mRNA spanned about 2.0 kb, and its expression was found only in the mammary epithelial cells. Glucocorticoid was essential for LAO gene expression. Thus, the LAO gene is expressed acutely upon the onset of milk synthesis. LAO mRNA increased 1 day before parturition, peaked during early to mid-lactation, and decreased at the end of lactation. This is the first demonstration showing that LAO is present in milk. Mastitis is caused by an intramammary bacterial infection. As mouse milk produced H 2 O 2 using endogenous free amino acids, we suggest that LAO, together with free amino acids, is responsible for killing bacteria in the mammary gland.

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 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.

To identify target cells of prolactin (PRL) in the male gonad, the expression of prolactin receptor (PRL-R) mRNA in adult rat testes was investigated by in situ hybridization using a digoxigenin-labeled cRNA probe. We also investigated PRL binding to testicular cells in vitro. Signals for PRL-R mRNA were detected not only in interstitial cells but also in spermatogenic cells. Although the reaction was positive in all phases of spermatogonia and spermatocytes, it disappeared in early round spermatids. No signals were detected in elongated spermatids or spermatozoa. The signal intensity varied among each phase of spermatogenic cells. PRL-R mRNA was expressed in all stages of the cycle of the seminiferous epithelium. PRL-R were detected on the surface of Leydig cells, Sertoli cells, all phases of spermatogonia and spermatocytes, elongated spermatids, and spermatozoa. Ovine PRL. did not bind to round spermatids. In Leydig cells, pachytene spermatocytes, and spermatozoa, PRL-R were observed in relatively large numbers. There were fewer receptors in other phases of spermatogenic cells. These results indicate that PRL-R mRNA expression is almost consistent with PRL binding sites except for elongated spermatids and spermatozoa, and suggest that PRL may have direct effects on spermatogenic cells.

To gain a better understanding of biochemical mechanisms of conceptus adhesion to the maternal endometrium in ruminant ungulates, the present study was performed to clarify roles of chemokines and extracellular matrix (ECM) components in the regulation of ovine blastocyst attachment to the endometrium. In addition to the chemokine, interferon-gamma inducible protein 10 kDa (IP-10, CXCL10), the chemokine receptor, CXCR3, also recognizes two other chemokines; monokine induced by IFN-gamma (MIG, CXCL9) and IFN-inducible T cell alpha chemoattractant (I-TAC, CXCL11). Similar to CXCL10, CXCL9, and CXCL11 were expressed in the uterus during the peri-implantation period, and CXCL9 mRNA expression was stimulated in endometrial explants from day 14 cyclic ewes by the addition of IFN-tau or IFN-gamma. Without ECM components, conceptus cell adhesion was low on day 14 of gestation and exhibited a 2.5-fold increase on day 17; adhesiveness on day 20 was 1/10 of that on day 14. Among various ECM components examined, trophoblast adhesion was greatest when fibronectin was used. Although day 14 conceptuses did not show much adhesive activity to fibronectin, day 17 trophoblast, and day 20 chorionic membrane exhibited 2.3-fold and 50-fold increase, respectively, which was enhanced by treatment with CXCL9 or CXCL10. These results indicate that through endometrial fibronectin and chemokines, ovine conceptus cells gain the ability to attach to the endometrium during pre-implantation period; however, elucidation of molecular mechanisms by which the conceptus acquires the adhesive ability during this time period awaits further investigation.