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Lysophosphatidic Acids Produced by Lysophospholipase D in MammalianSerum and Body Fluid

AKIRA TOKUMURA,a,c SHUJI YAMANO,b TOSHIHIRO AONO,b

AND KENJI FUKUZAWAa

aFaculty of Pharmaceutical Sciences, The University of Tokushima,1-78 Shomachi, Tokushima 770-8505, JapanbSchool of Medicine, The University of Tokushima, 3-18-15 Kuramoto-cho,Tokushima 770-8503, Japan

It has long been known that incubated mammalian plasma and serum contain avasoactive phospholipid, later identified as lysophosphatidic acid (LPA).1 We previ-ously found that metal-ion-dependent lysophospholipase D (LPLD) is involved inthe accumulation of LPAs in incubated rat plasma; the enzyme preferentially hydro-lyzed unsaturated lysophosphatidylcholines (LPCs) to saturated LPCs in the plas-ma.1,2 The plasma LPLD has ultimate physiological significance through supplyingbioactive LPA continuously to peripheral tissues. In this study, we found that theLPLD activity in human serum is increased during pregnancy, and that the sameenzyme activity is distributed in blood-borne follicular fluid collected from womenfor in vitro fertilization treatment. These results suggest the physiological signifi-cance of LPAs produced by the extracellular LPLD on reproductive biology. In linewith this suggestion, we found that LPA promotes maturation of oocytes and theirtransport through the oviduct.

METHODS

LPLD activities in human serum, plasma, and follicular fluids were measured es-sentially as described previously for those in rat plasma.2 Briefly, body fluids wereincubated with 0.1 volume of a solution of 1-[palmitoyl-1-14C]-LPC (7 nmol/mL) insaline containing 0.25% BSA at 37°C for 6 h. At 0, 2, 4, and 6 h, 0.2-mL aliquotswere diluted to 2 mL with 2% KCl. Lipids were extracted by the method of Blighand Dyer after acidification of samples (pH 2.5) and separated by TLC with chloro-form/methanol/20% ammonium hydroxide (60:35:8) together with carrier LPA(0.1 µmol). The percentage conversion of [14C]LPC to LPA were calculated fromthe radioactivities of bands on the TLC plates and expressed in terms of percentageper hour.

Excised oviducts of ICR-strain mice were placed in 1 mL of basic culture medi-um (Whittingham’s T6 medium supplemented with 0.4% fatty-acid-free BSA) with

cCorresponding author. Voice: 81-88633-7249; fax: 81-88633-7248.tokumura@ph.tokushima-u.ac.jp

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or without LPA and incubated at 37°C under 5% CO2 in humidified air for 24 h.After incubation, the number of ova flushed out of the oviduct was first counted.Next, a fine glass pipette was cannulated to the ampulla of the oviduct and flushedwith basic culture medium. The number of ova flushed out of the oviduct was count-ed and defined as the number of ova residing in the oviduct.

Cumulus oocyte complexes were collected from the ovaries of golden hamsters48 h after intraperitoneal injection of pregnant mare’s serum gonadotropin (20 IU),and incubated with or without LPA. After incubation, the oocytes were freed fromcumulus cells and observed under an inverted phase-contrast microscope to evaluatewhether they reached to metaphase II or not. In some experiments, the cumulus cellswere removed from the oocytes by treatment with hyaluronidase at 37°C, and thedenuded oocytes were also treated with or without LPA.

RESULTS AND DISCUSSION

We attempted to assess the initial rate of LPA accumulation by measuring the per-centage conversion of [14C]LPC added to human serum, plasma, or follicular fluid.Radioactive LPC was found to be converted to LPA by LPLD at a constant rate with-in 6 h. Values for LPA generation in incubated follicular fluids from five women pro-grammed for in vitro fertilization were similar, and variation in the values for folli-cular fluids from the same patient were very low [0.99 ± 0.02 (n = 6), 0.72 ± 0.03(3), 0.81 ± 0.03 (3), 0.65 ± 0.02 (3), 0.87 ± 0.01 %/h (3)]. The LPLD activity of se-rum of the patients (0.48 ± 0.06 %/h) was about 2.5-fold that of healthy women (0.19± 0.01%/h, n = 5). These results suggest the pathophysiological significance of en-hanced production of LPA in the blood and blood-derived fluids of women after ova-rian hyperstimulation.

FIGURE 1 shows results on LPLD activity of serum from pregnant and plasmafrom nonpregnant women. The LPLD activity in the first trimester of pregnancy was

FIGURE 1. LPLD activity of serum from pregnant and plasma from nonpregnant wom-en. Numbers in parentheses show numbers of serum preparation.

349TOKUMURA et al.: LPA

significantly higher than that of nonpregnant women. Interestingly, there was a fur-ther increase in the LPLD activity in the third trimester. These results suggest thatLPA produced by serum LPLD has a physiological role in maintenance of pregnancyand induction of delivery. It should be mentioned that LPA is known to contract uter-ine smooth muscles in vivo and in vitro.3

As described in the preceding, we found that LPA is generated in incubated fol-licular fluid by the action of LPLD, as occurs in human serum. Oocyte-cumulus cellmatrix is drenched with a considerable amount of follicular fluid, and thus LPA mayplay an important role in transport and maturation of oocytes. Therefore, in thisstudy, we first examined the effects of LPA on ovum transport in mouse oviducts.The ovum transport rate after addition of 10 µM LPA was significantly higher thanthat of the control (60.4 ± 9.5% vs. 33.1 ± 7.6%, P < 0.05), although addition of 1µM LPA slightly, but not significantly, increased the ovum transport (42.5 ± 9.2µM). Pretreatment of ova with 200 ng/mL of pertussis toxin (PT) suppressed the in-crease in ovum transport rate that was stimulated by 10 µM LPA, whereasindomethacin at 10 µM did not inhibit the transport rate (FIG. 2). In addition, theovum transport rate in the LPA + verapamil group was significantly lower than thatin the LPA group (FIG. 2). These results indicate that the accelerating effect of LPAon mouse ovum transport is probably dependent on oviduct smooth muscle contrac-tion via a voltage-sensitive calcium channel mediated by a PT-sensitive G-protein-linked receptor.

Next, we examined whether LPA accelerates nuclear maturation of hamsteroocytes. The maturation rate of oocytes incubated with 10 µM LPA (74%) was sig-nificantly higher than that of control (63%). The denudation of cumulus oocyte com-plex resulted in reduction in the maturation rate (46%), and there was no significantincrease in the maturation rate of the denuded oocytes incubated with LPA (44%). Arecent study showed that suppression of gap-junctional communication betweencumulus cells progressed oocyte nuclear stage.4 In addition, LPA has been shown to

FIGURE 2. Defects of indomethacin, PT and verapamil on the mouse ovum transportaccelerated by LPA. Excised oviducts were placed in 1 mL of basic culture medium with andwithout 10 µM LPA in the presence or absence of 10 µM indomethacin or verapamil. Theoviducts were also preincunated with PT (200 ng/mL) for 6 h before transferring to the cul-ture medium with or without LPA. *P < 0.05 versus untreated.

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close gap junctional communication in rat-liver cell.5 Taken altogether, it can bespeculated that LPA promotes oocyte nuclear maturation by inhibiting the gap junc-tional communication.

In conclusion, LPAs generated by extracellular LPLD can play an importantphysiological role in the reproductive biology of mammals.

REFERENCES

1. TOKUMURA, A., et al. 1986. Involvement of lysophospholipase D in the production oflysophosphatidic acid in rat plasma. Biochim. Biophys. Acta 875: 31–38.

2. TOKUMURA, A., et al. 1998. Metal-ion stimulation and inhibition of lysophospholipase Dwhich generates bioactive lysophosphatidic acid in rat plasma. Lipids 33: 751–756.

3. TOKUMURA, A., et al. 1980. Stimulatory effects of lysophosphatidic acids on uterinesmooth muscle of non-pregnant rats. Arch. Int. Pharmacodyn. Therap. 245: 74–83.

4. HII, C.S.T., et al. 1994. Lysophosphatidic acid inhibits gap-junctional communicationand stimulates phosphorylation of the gap junction protein connexin-43 in WB cells:possible involvement of the mitogen-activated protein kinase cascade. Biochem. J.303: 475–477.

5. SHIMADA, M. & T. TERADA. 1999. Phosphorylation of connexin-43, gap-junctional pro-tein, in cumulus cells is regulated by mitogen-activated protein kinase and phosphati-dylinositol 3-kinase during in vitro meiotic resumption in porcine follicular oocytes.J. Mamm. Ova Res. 16: 37–42.