Shuji Yamano

Increased Production of Bioactive Lysophosphatidic Acid by Serum Lysophospholipase D in Human Pregnancy

Abstract Lysophosphatidic acid (LPA) is a prototype of the lysophospholipid mediator family and has multiple effects in the female reproductive system. Although several metabolic routes have been reported for intracellular formation of LPA, a unique route involving lysophospholipase D, an extracellular enzyme that produces LPA in blood and body fluids, is particularly intriguing for its agonistic role. In this study, using an assay with radioactive palmitoyl-lysophosphatidylcholine, we found that lysophospholipase D activity producing palmitoyl-LPA in human serum gradually increased during pregnancy. Elevated activity of lysophospholipase D was not caused by changes in levels of their precursors, lysophosphatidylcholines, in nonpregnant women or in pregnant women at different gestational periods. With increasing length of gestation, the elevated activity in pregnant women was found to produce increasing proportions of LPA with a palmitoyl group versus other LPAs. These results suggest that LPA formed by increased activity of lysophospholipase D in blood might participate in maintenance of pregnancy.

EFFECT OF LYSOPHOSPHATIDIC ACID ON THE PREIMPLANTATION DEVELOPMENT OF MOUSE EMBRYOS

The effect of lysophosphatidic acid (LPA) on the preimplantation development of mouse embryos was examined. The blastocyst rates from 2‐cell and 4‐cell stage embryos were significantly higher in the presence of LPA at the pronuclear stage. However, the blastocyst rate from 4‐cell stage embryos was significantly lower when LPA was added to the medium at the 4‐cell stage than when it was added at the pronuclear stage. Furthermore, pretreatment of pronuclear stage embryos with pertussis toxin suppressed LPA‐induced embryo development, suggesting that it is probably mediated by a Gi‐protein‐linked receptor.

Effect of activation with Ca ionophore A23187 and puromycin on the development of human oocytes that failed to fertilize after intracytoplasmic sperm injection

OBJECTIVE To determine the effect of sequential oocyte activation with calcium ionophore A23187 (A23187) and puromycin on oocytes that were unfertilized after intracytoplasmic sperm injection (ICSI). DESIGN Laboratory examination. SETTING The in vitro fertilization/embryo transfer unit in Tokushima University Hospital. PATIENT(S) Discarded unfertilized oocytes following ICSI. INTERVENTION(S) All 172 oocytes that showed no evidence of normal fertilization 18 hours after ICSI were exposed to A23187 (5 microM) for 5 minutes and consequently were treated with puromycin (10 microg/mL) for 5 hours. MAIN OUTCOME MEASURE(S) The activation rate, proportion of oocytes that showed two pronuclei with the second polar body (2PN2PB), and cleavage rate were calculated. Chromosomal analysis of the oocytes with 2PN2PB was also performed. RESULT(S) The treatment activated 146 out of 172 oocytes (84.9%) and 30.1% of the activated oocytes showed 2PN2PB. Sixteen of 25 oocytes with 2PN2PB (64.0%) cleaved. There was no difference in the activation rate, proportion of activated oocytes with 2PN2PB, or cleavage rate between oocytes that were injected with a motile spermatozoon and those injected with an immotile spermatozoon. Chromosomal analysis showed a normal diploid set of chromosomes (n = 46) in four of five analyzable oocytes. CONCLUSION(S) The sequential treatment of calcium ionophore A23187 and puromycin activates unfertilized oocytes after ICSI. The resultant oocytes with 2PN2PB can cleave.

Postmenopausal changes in production of type 1 and type 2 cytokines and the effects of hormone replacement therapy.

ObjectiveAn appropriate defense against infective agents or malignant cells is attributed to the exquisitely balanced T helper 1 type (cellular) and T helper 2 type (humoral) immune reactions. We investigated the effect of hormone replacement therapy (HRT) on postmenopausal changes in the production of interferon (IFN)-&ggr; and interleukin (IL)-10, a type 1 and a type 2 cytokine, respectively. DesignBoth cytokines were measured by ELISA in the supernatant of lipopolysaccharide-stimulated whole blood cells from 72 untreated and 44 HRT-treated women. Thirteen women were examined before and during HRT. ResultsThe production of IFN-&ggr; in women in their 40s and in postmenopausal women was significantly higher compared with that of younger women. However, IFN-&ggr; fell to the lowest level in the late postmenopausal stage, whereas the production of IL-10 increased gradually with age and in parallel with the postmenopausal period. Thus, in women in the mid-and late postmenopausal period, excessive production of type 2 cytokine (IL-10) compared with type 1 cytokine (IFN-&ggr;) occurred. The IFN-&ggr; levels of women on HRT were significantly lower than those of untreated women in the early and mid-postmenopausal stages, and IL-10 levels of women on HRT were significantly lower than those of untreated women in the mid-and late postmenopausal stages. HRT induced a significant decrease in the production of IL-10 and tended to lower the level of IFN-&ggr;. ConclusionsProduction of IL-10 is augmented in postmenopausal women. HRT probably prevents postmenopausal women from an aberration of the immune system by improving the balance of type 1 and type 2 immune reactions.

Transient Increase in the Levels of T-Helper 1 Cytokines in Postmenopausal Women and the Effects of Hormone Replacement Therapy

The aim of this study was to determine, at least in part, T-cell function in postmenopausal women and the effects of hormone replacement therapy (HRT). Levels of T-helper 1 (Th1) cytokines (IL-2 and IFN-γ) and T-helper 2 (Th2) cytokines (IL-4 and IL-10) produced by phytohemagglutinin-stimulated whole blood cells from 72 untreated and 44 HRT-treated women were measured by ELISA. Thirteen of the 44 HRT-treated women were examined before and during HRT. The production of IL-2 increased gradually with advance of the postmenopausal period. The levels of IL-2 in women in the early (≤10 years) and mid (>10 and <30 years) postmenopausal stages were significantly higher than those in women in their second, third and fourth decades. The level in women in the late (≧30 years) postmenopausal stage, however, was significantly lower than those in women in the early and mid postmenopausal stages. The level of IFN-γ was highest in women in the mid postmenopausal stage. On the other hand, the levels of Th2 cytokines did not change with age or after menopause until the mid postmenopausal period but were significantly lower in women in the late postmenopausal stage. IFN-γ levels in women on HRT were significantly lower than those in untreated postmenopausal women at all postmenopausal stages. HRT induced a significant decrease in the production of IL-2 and IL-4. In conclusion, production of Th1 cytokines is augmented in women after menopause. HRT prevents this increase, thereby improving the aberration of Th1/Th2 balance that is implicated in an inadequate immune response and pathological conditions.

A combination of calcium ionophore and puromycin effectively produces human parthenogenones with one haploid pronucleus.

Parthenogenetic activation with various combinations of the calcium ionophore A23187 and protein synthesis or phosphorylation inhibitors was investigated as a means of producing human parthenogenones with one haploid pronucleus. Unfertilised human aged oocytes exposed to 5 microM A23187 for 5 min were treated with 10 microg/ml puromycin (puromycin group, 46 oocytes) or 2 mM 6-dimethylaminopurine (DMAP group, 42 oocytes) for 5 h. Oocytes treated only with A23187 served as a control (control group, 40 oocytes). After washing the oocytes, they were incubated for up to 37 h. Evidence of activation (pronuclear formation) and cleavage was observed 18 h and 42 h after A23187 treatment, respectively. Activation rates in the puromycin and DMAP groups were significantly higher than in the control group (91% (42/46) and 77% (34/44) vs 20% (8/40), p < 0.05, respectively). In the puromycin group, 81% (34/42) of the activated oocytes showed one pronucleus with the second polar body (2ndPB), whereas none (0/34) of the activated oocytes in the DMAP group extruded the 2ndPB. The cleavage rate in the puromycin group was significantly lower than in the DMAP group (38% vs 68%, p < 0.05). The activated oocytes which had one pronucleus with the 2ndPB in the puromycin group showed a haploid set of chromosomes (10/13). In conclusion, the combination of A23187 and puromycin is effective for producing human parthenogenones with one haploid pronucleus.

Effect of lysophosphatidic acid on the ovum transport in mouse oviducts.

The effects of lysophosphatidic acid (LPA) on ovum transport in mouse oviducts were studied. When excised oviducts were incubated at 37 degrees C under 5% CO2 in humidified air for 24 hours, addition of LPA at 10 microM to the medium significantly accelerated the rate of ovum transport, and 1 microM LPA slightly increased the ovum transport rate. These increases were not inhibited by 10 microM indomethacin, a cyclooxygense inhibitor, but were suppressed by 260 ng/ml of pertussis toxin or 10 microM verapamil, a voltage-sensitive calcium channel blocker. These data suggested that LPA stimulates mouse ovum transport by contracting oviductual smooth muscle via a voltage-sensitive calcium channel mediated by a pertussis toxin-sensitive G-protein-linked receptor.

Lysophosphatidic acids produced by lysophospholipase D in mammalian serum and body fluid.

It has long been known that incubated mammalian plasma and serum contain a vasoactive phospholipid, later identified as lysophosphatidic acid (LPA).1 We previously found that metal-ion-dependent lysophospholipase D (LPLD) is involved in the accumulation of LPAs in incubated rat plasma; the enzyme preferentially hydrolyzed unsaturated lysophosphatidylcholines (LPCs) to saturated LPCs in the plasma.1,2 The plasma LPLD has ultimate physiological significance through supplying bioactive LPA continuously to peripheral tissues. In this study, we found that the LPLD activity in human serum is increased during pregnancy, and that the same enzyme activity is distributed in blood-borne follicular fluid collected from women for in vitro fertilization treatment. These results suggest the physiological significance of LPAs produced by the extracellular LPLD on reproductive biology. In line with this suggestion, we found that LPA promotes maturation of oocytes and their transport through the oviduct.

Lysophosphatidic acid stimulates nuclear and cytoplasmic maturation of golden hamster immature oocytes in vitro via cumulus cells.

Lysophosphatidic acid (LPA), a member of the phospholipid autacoid family, is induced in incubated human follicular fluid by lysophospholipase D. It is well known that LPA functions as a growth factor and the hypothesis that LPA in human follicular fluid takes a part in meiosis of oocytes is quite plausible. We studied the effects of LPA on the maturation of golden hamster immature oocytes in vitro. Hamster oocytes with a germinal vesicle were cultured in Tyrodes albumin lactate pyruvate (TALP) medium with 10(-5) M LPA, 10 ng/ml epidermal growth factor (EGF), 30 ng/ml insulin-like growth factor-1, 1 ng/ml tumor growth factor-alpha or 1 ng/ml basic fibroblast growth factor. The nuclear maturation rates in the LPA and EGF groups were significantly higher than in the control group and the other growth factors did not show any stimulatory effect (LPA group; 74.3% [75/101], EGF group; 82.4% [89/108] vs. control group; 60.2% [59/98], p < 0.05, p < 0.01, respectively). When the cells of cumulus were removed, EGF and LPA did not increase the nuclear maturation rates. Cotreatment EGF and LPA did not significantly enhance the stimulatory effect observed with LPA alone on maturation in vitro. The penetration rate determined by the zona-free hamster oocyte test was significantly higher in the LPA group than in the control group (26.7% vs. 13.2%, p < 0.05) and was comparable with that of oocytes matured in vivo. In conclusion, LPA stimulates the nuclear and cytoplasmic maturation of hamster immature oocytes via cumulus cells.

Relationship among serum levels of luteinizing hormone, estradiol and progesterone during follicle stimulation and results of in vitro fertilization and embryo transfer (IVF-ET)

Eighty-eight IVF-ET cycles were classified into four groups according to the results of IVF-ET (Group A—conceptional cycles, 10 cycles; Group B—cycles with cleaved oocytes, 58 cycles; Group C—cycles with fertilized oocytes, 9 cycles; Group D—cycles without fertilization, 11 cycles). Serum luteinizing hormone (LH), estradiol (E2), and progesterone (P) levels during follicle stimulation were studied in these groups. Patients participated in our IVF-ET program due to irreparable tubal damage. Follicle development was stimulated with a clomiphene—human menopausal gonadotropin (hMG)—human chorionic gonadotropin (hCG) regimen. Group C showed a low E2response to follicle stimulation. Groups B and D showed significantly higher serum P levels on day 0 (the day of hCG injection) than Group A (Group A, 0.73 ± 0.11, vs Groups B and D, 1.43 ± 0.15 and 2.17 ± 0.42 ng/ml; P <0.01). The effects of serum P and LH levels on the fertilization and pregnancy rates were studied. The pregnancy rate was not affected by the serum LH level but was only 2.7% in cycles in which serum P was 1.2 ng/ml on day 0, which was significantly lower than that in cycles in which serum P was <1.2 ng/ml on day 0 (19.1%) (P <0.05). The fertilization rate was significantly lower in the cycles with higher levels of serum P and/or LH than in cycles in which serum P was <1.2 ng/ml and serum LH was normal (50.5 vs 78.8%; P <0.01). These findings suggest that the serum P level, but not the LH level, during follicle stimulation is closely related to the achievement of pregnancy.