Hiroyuki Minoura

Ornithodoros moubata: host immunoglobulin G in tick hemolymph.

Hemolymph proteins of a soft tick, Ornithodoros moubata, were analyzed immunochemically and biochemically. The components of tick hemolymph proteins were shown to be totally different from the host (rabbit) serum proteins by polyacrylamide gel electrophoresis with sodium dodecyl sulfate and Coomassie blue or silver stain. However, in the hemolymph of ticks engorged from rabbits immunoglobulin G was detected by immunoblotting analysis with goat anti-rabbit immunoglobulin G. The concentration of rabbit Immunoglobulin G in tick hemolymph changed with the physiological stages after a blood meal. Immunoglobulin G was isolated from tick hemolymph by affinity chromatography on a Protein A-Sepharose 4B column. Analysis of the isolated immunoglobulin G from tick hemolymph with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Ouchterlony double diffusion test showed it to be composed of the same subunits as heavy and light chains of host (rabbit) immunoglobulin G. Tracer experiments showed that 125I-labeled heavy and light chains of immunoglobulin G were detected in an intact form in hemolymph from ticks that sucked 125I-labeled rabbit immunoglobulin G through an artificial membrane. These facts suggested that the host rabbit immunoglobulin G ingested in the tick midgut passed through the gut wall without digestion. By solid-phase enzyme immunoassay, immunoglobulin in the hemolymph was shown to retain its antibody activity.

Host immunoglobulin G titre and antibody activity in haemolymph of the tick, Ornithodoros moubata

Abstract. Immunoglobulin G (IgG) in tick haemolymph was analysed immunochemically and biochemically for its antigenicity, antibody activity and relative concentration in a soft tick, Ornithodoros moubata (Murray) sensu Walton 1962 (Acari: Argasidae). Ouchterlony immunodiffusion tests showed that haemolymph from a tick engorged on rabbit IgG (or human IgG) through an artificial membrane, reacted with anti‐rabbit IgG (anti‐human IgG) but not with anti‐human IgG (anti‐rabbit IgG). This indicates that haemolymph of the fed tick contains IgG with a similar antigen specificity to host blood IgG. IgG from tick haemolymph was demonstrated by enzyme immunoassay to have the same antibody activity as ingested IgG. The IgG concentration in tick haemolymph was measured by a quantitative single immunodiffusion test. Changes of IgG titre after a bloodmeal were correlated with IgG activity, which was low for 5 days after a bloodmeal and then suddenly increased. The IgG titre reached a maximum 7 days post‐engorgement, and remained high for over 4 months during and after oviposition. 125I‐labelled IgG was injected into the tick haemocoel to determine the persistence of IgG in the haemolymph. Recovery of labelled IgG was low at 1 and 3 days, and high at 5, 8 and 16 days after engorgement. The data suggest that IgG in haemolymph disappears quickly soon after engorgement possibly by degradation and/or absorption (adhesion to tissues).

Expression of glucose transporter 4 mrna in adipose tissue- and skeletal muscle of ovariectomized rats treated with sex steroid hormones

The effects of 17beta-estradiol (E) and/or progesterone (P) on glucose transporter 4 (GLUT4) expression in the adipose tissue and skeletal muscle of ovariectomized female rats were studied. The Sprague-Dawley rats received daily subcutaneous injections of various doses of E and/or P for 7 days (n=5-6 per dose). The expression of GLUT4 mRNA was assessed by performing ribonuclease protection assays. GLUT4 protein levels were assessed by Western blotting assays. The adipose tissue levels of GLUT4 mRNA were reduced by the administration of 50 microg E, which resulted in unphysiologically high serum E concentrations. Although the GLUT4 mRNA levels did not change after the administration of 10 microg E or 5 mg P, they were reduced significantly to approximately half the control group level by the administration of both hormones (p <0.01). The skeletal muscle GLUT4 mRNA levels were not changed significantly by hormone treatment. These findings suggest that E and P may be involved in the regulation of GLUT4 mRNA expression in adipose tissue.

Expression of Tenascin-C in Stromal Cells of the Murine Uterus During Early Pregnancy: Induction by Interleukin-1α, Prostaglandin E2, and Prostaglandin F2α

Abstract Tenascin-C (TN-C), an extracellular matrix glycoprotein, is known to be expressed in uterine stroma in the peri-implantation period. Examination of the spatiotemporal pattern during early pregnancy using immunohistochemistry and in situ hybridization revealed TN-C expression in the stroma beneath the luminal epithelia of the murine endometrium on Days 0 and 1 of pregnancy, subsequent disappearance, and reappearance on Day 4. After decidualization, tissue around the deciduoma was positive. In situ hybridization demonstrated TN-C production by the stromal cells adjacent to the epithelia. To investigate the regulation of TN-C expression in vitro, murine uterine stromal and epithelial cells were isolated and cultured. Addition of interleukin-1α (IL-1α) and prostaglandin E2 (PGE2) and F2α (PGF2α) induced TN-C expression in the stromal cells at both protein and mRNA levels, while the sex steroid hormones, progesterone and β-estradiol, exerted little effect. Immunohistochemistry using anti-IL-1α antibody showed epithelial cells to be positive on Days 2–4 of pregnancy, and addition of progesterone but not β-estradiol enhanced IL-1α expression in epithelial cells in vitro. In a culture insert system, TN-C expression by stromal cells cocultured with epithelial cells was induced by addition of progesterone alone that was blocked by additions of anti-IL-1α antibody. Collectively, these findings indicate that TN-C expression in the preimplantation period is under the control of progesterone, but not directly, possibly by the paracrine and autocrine intervention of IL-1α secreted by epithelial cells and PGE2 and PGF2α secreted by stromal cells.

Possible direct effect of gonadotropin releasing hormone on human endometrium and decidua.

Decidualization of endometrial tissues, which is essential for implantation and the continuation of pregnancy, is induced by pituitary hormones that are regulated by gonadotropin releasing hormone (GnRH). Our objective was to determine the role of a direct action of GnRH on endometrial tissues by comparing the characteristics of receptors for GnRH in human endometrial and decidual tissues. Competitive binding studies were performed with the protease-resistant GnRH analogues, buserelin and [125I] buserelin. The effects of buserelin on phosphoinositol turnover were determined by the measurement of inositol 1,4,5-triphosphate(IP3). The values for the dissociation constant (Kd) and number of binding sites (Bmax) per unit protein versus buserelin for endometrial tissues did not differ from the values for decidual tissues. However, the Bmax per unit DNA was significantly higher in endometrial tissues. Also, buserelin induced a significant increase in IP3 in decidual tissue. These results indicate that GnRH may be a potential modulator of the function in human endometrium and decidua. The signal transduction mechanism for GnRH action appeared to involve the accelerated turnover of phosphoinositol.

A novel cDNA clone encoding a prolactin-like protein that lacks the two C-terminal cysteine residues isolated from bovine placenta

A new prolactin-like cDNA clone, bPLP-IV, was isolated from a bovine placental cDNA library and the complete nucleotide sequence was determined. The bPLP-IV encodes a protein consisting of 237 amino acids, which is related to, but different from seven other known bovine prolactin-like proteins including two placental lactogens. The predicted amino acid sequence of the bPLP-IV shows over 52% identity to other known members of bovine prolactin-like proteins, 48% to bovine prolactin, 40% to both two bovine placental lactogens and only 22% to bovine growth hormone. The bPLP-IV protein has a unique feature in its primary structure, lacking the two C-terminal cysteine residues which are completely conserved in all other known members of prolactin-growth hormone-placental lactogen gene family. The expression of bPLP-IV in developing bovine placenta was apparently stage-specific, being maximal in the full-term placenta.

Expression of matrix metalloproteinase-3 in mouse endometrial stromal cells during early pregnancy: Regulation by interleukin-1α and tenascin-C

During the peri-implantation period, the endometrium undergoes tissue remodeling and cellular rearrangement. To clarify the involvement of matrix metalloproteinases (MMPs) in endometrial remodeling, we isolated total RNAs from the endometrium of non-pregnant and pregnant mice on days 3 to 5 and evaluated mRNA expression of MMP-2, -3, -9, -11 and -13 using reverse transcription–polymerase chain reaction (PCR). Prompt increases in MMP-3 and -13 mRNA were found on day 4 of pregnancy. Quantitative real-time PCR showed that expression of MMP-3 and -13 increased significantly on day 4, up to 8.4 ± 2.7 times and 3.4 ± 1.5 times, respectively, the level in non-pregnant endometrium (p < 0.05). On day 4, immunohistochemistry demonstrated MMP-3-positive endometrial stromal cells. At the same time, tenascin-C (TN-C) mRNA increased 11.1 ± 4.0 times from the level in non-pregnant endometrium (p < 0.004). To clarify regulation of MMP-3 expression, we examined the effects of interleukin-1α (IL-1α) and TN-C on MMP-3 mRNA in cultured mouse endometrial stromal cells. Both substances resulted in a dose-dependent increase in MMP-3 mRNA (6.1 ± 1.8-fold at 1 ng/ml of IL-1α and 3.9 ± 1.8-fold at 10 μg/ml of TN-C). This study shows that MMP-3 expression is upregulated in endometrial stromal cells of the peri-implantation period and may be controlled by IL-1α and TN-C.

Comparison of Piezo-Assisted Micromanipulation with Conventional Micromanipulation for Intracytoplasmic Sperm Injection into Human Oocytes

Objective: To compare the efficacy of piezo-assisted micromanipulation with conventional micromanipulation for intracytoplasmic sperm injection (ICSI) into oocytes in patients with impaired semen parameters and no success with in vitro fertilization (IVF). Study Design: A retrospective randomized study was conducted on 204 cycles for 104 couples with piezo-assisted ICSI and 122 cycles for 96 couples with conventional ICSI. Piezo-assisted ICSI consists of two steps, namely penetration of the zona pellucida alone with a piezo-pulse and then puncturing of the oolemma with a light negative pressure without piezo, as with conventional ICSI. The tips of injection pipettes were prepared after pulling by breakage with a scalpel under a microscope, so that the inner diameter at and near the tip was 5 µm, as for conventional ICSI. Results: Piezo-assisted ICSI demonstrated significantly more favorable results, with a fertilization rate of 90.3% (conventional ICSI: 83.1%, p < 0.01) and a cleavage rate of 88.1% (conventional ICSI: 84.6%, p < 0.01). Conclusion: Piezo-assisted ICSI is easy to incorporate a spermatozoa exactly into the ooplasm with little deformation of the oocyte during insertion. Piezo-assisted ICSI can be used effectively for human oocytes to improve the fertilization, cleavage rates.

Effects of epidermal growth factor on preimplantation mouse embryos

Purpose:Our purpose was to clarify the effects of epidermal growth factor (EGF) on mouse preimplantation embryos.Methods:We examined the effect of EGF on two-cell and four-cell stage mouse embryos cultured in vitro. In preimplantation embryos, we analyzed the binding of125I-EGF by autoradiography and EGF receptor mRNA by the reverse transcription-polymerase chain reaction.Results:At more than 0.005 ng/ml, EGF relieved the two-cell block and regulated the differentiation of morula-stage embryos. These effects were negated by antiserum. EGF did not exhibit any marked effect on embryos between the four-cell and the morula stages. Specific binding for EGF and EGF receptor mRNA was detected during and after the morula stage.Conclusions:The effects of EGF on preimplantation mouse embryos differed according to the stage of development, promoting cleavage before the four-cell stage and regulating differentiation after the morula stage. This regulatory action is thought to be transmitted to the cells via EGF receptors.

Intrinsic Ureteric Involvement by Endometriosis: A Case Report

Endometriosis occasionally involves the urinary tract, and a ureteral obstruction from this order constitues a rare variant with serious consequences. Intrinsic ureteric involvement by endometriosis is an exceedingly rare event. This case report describes intrinsic ureteric involvement by endometriosis. The case involved 47‐year‐old woman, gravida 4, para 2, who had a 4‐year history of dysmenorrhea and hypermenorrhea. An intravenous pyelogram showed a right hydronephrosis. She underwent a total abdominal hysterectomy and a right ureteroureterostomy. A pathologic examination revealed complete obstruction of the right ureter by intrinsic intramural endometriosis. We conclude that because ureteral endometriosis, especially intrinsic endometriosis, is usually silent and results in a high rate of renal loss before recognition, physicians should have a hightened awareness of this uncommon but serious manifestation of endometriosis.