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Biology of Reproduction 61, 195-199 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Articles

Production of Lysophosphatidic Acids by Lysophospholipase D in Human Follicular Fluids of In Vitro Fertilization Patients1

Akira Tokumura2,a, Maki Miyakea, Yuko Nishiokaa, Shuji Yamanob, Toshihiro Aonob, and Kenji Fukuzawaa

a Faculty of Pharmaceutical Sciences and b Department of Obstetrics and Gynecology, School of Medicine, The University of Tokushima, Tokushima 770–8505, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Lysophosphatidic acids (LPAs) are known to be normal constituents of mammalian serum, and they mimic some biological effects of the serum. We previously reported that lysophospholipase D (LPLD) was involved in the accumulation of LPAs in incubated rat plasma and serum. In this study we detected, by gas-liquid chromatography, various molecular species of LPA in follicular fluids collected from women programmed for in vitro fertilization. When the follicular fluid was incubated at 37°C for 48 h, persistent increases in the amounts of LPAs were observed concomitant with decreases in the amounts of the corresponding lysophosphatidylcholines (LPCs), although the concentrations of saturated LPCs increased in the first 6 h of incubation. These results suggest that human follicular fluid has LPLD activity, and this was confirmed by experiments with follicular fluids mixed with an exogenous radioactive LPC. The LPLD showed preference for unsaturated over saturated LPCs, similar to plasma LPLD, indicating that it originated from the circulation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Lysophosphatidic acids (LPAs) are important members of the phospholipid autacoid family, and their growth factor-like and hormone-like activities have attracted attention [1, 2]. LPA receptors have been characterized by photobinding [3] and radiobinding studies [4, 5] and have been confirmed by molecular cloning of at least three distinct types of encoding genes [6–8]. LPAs are known to be present at micromolar concentrations in freshly prepared mammalian serum [9] and to mimic the effects of the serum on various cells [1, 2]. Activated platelets have been reported to release LPA into the circulation [9], and it has been suggested that two mechanisms involving hydrolysis of phosphatidic acid by phospholipase A2 (or A1) or phosphorylation of monoacylglycerols participate in the accumulation of micromolar concentrations of LPA in serum [9, 10]. On the other hand, it has long been known that incubated mammalian plasma and serum contain a vasoactive phospholipid, later identified as LPA [1]. The concentration of LPA in incubated rat plasma was reported to reach nearly 70 µM [11], and it was suggested that it was produced from plasma lysophosphatidylcholine (LPC) by metal ion-dependent lysophospholipase D (LPLD) [12]. From these results we expected that LPA could be produced in body fluids by the same mechanism as that reported for LPA production in rat plasma.

At the time of ovulation, follicular fluid accompanies development of the female gamete and may participate in fertilization and embryonic development. Therefore, follicular fluid is often used as a supplement in culture media for in vitro human fertilization [13]. Ovarian follicular fluid is secreted from thecal blood vessels and is modified by granulosa cells [14]. The follicular fluid contains high concentrations of growth factors and steroid hormones [14] and may contain other unknown components. LPAs are known to activate Ca2+-dependent opening of Cl- channels in Xenopus oocytes [15–18], suggesting their physiological significance in egg maturation. We have reported that addition of LPA to culture media stimulated preimplantation development of mouse embryos in vitro [19]. There is a recent report that bovine luteal cells possess functional receptors for LPAs and that LPAs up-regulate forskolin- and luteotropic hormone/guanosine 5'-triphosphate (GTP)-induced adenylate cyclase activity [5], indicating other, as yet unidentified, physiological roles of LPAs in the ovary besides egg maturation. These findings prompted us to examine whether LPAs are present in fresh and incubated human follicular fluids and, if so, how they are accumulated.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Collection of Follicular Fluid

Five infertile patients who received in vitro-fertilized embryos at Tokushima University Hospital were involved in this study. The Ethics Committee of the University of Tokushima approved the in vitro fertilization-embryo transfer (IVF-ET) treatment in our hospital. Informed consent for experimental use of follicular fluids was obtained from all patients. The mean age of patients was 33.6 ± 2.2 yr, and the duration of infertility was 7.6 ± 1.0 yr. Indications for IVF-ET treatments were as follows: one with tubal blockage (patient 4), one with tubal blockage and endometriosis (patient 5), two with severe oligozoospermia (patients 1 and 3), and one with antisperm antibody (patient 2). The patient with tubal blockage and endometriosis became pregnant during the IVF-ET treatment cycle. Ovarian stimulation was performed as we reported previously [20]. Administration of 600 µg/day of the GnRH agonist buserelin (Suprecur; Hoechst-Marion-Roussel Co., Tokyo, Japan) was started from the midluteal phase of the treatment cycle for pituitary desensitization. After menstruation, 225 IU/day of FSH (Fertinorm P; Serono Japan Co., Tokyo, Japan) was given for 5 days, beginning 12 days before oocyte retrieval. Consecutively, 150 IU of human menopausal gonadotropin (Humegon; N.V. Organon, Tokyo, Japan) was given daily for 4 days. A dose of 5000 IU of hCG (Gonatropin; Teikoku Hormone Mfg. Co., Tokyo, Japan) was administered 35 h before oocyte retrieval. Oocytes were retrieved transvaginally using a needle-guided technique and ultrasound. Follicles of more than 16-mm diameter were aspirated, and follicular fluids with a mature oocyte and no blood contamination were centrifuged at 1000 x g for 15 min at 4°C to eliminate residual cells. The follicular fluids were stored at -20°C and were assayed within 1 wk.

Preparation of Serum

Blood samples were collected in tubes (Vacutainer + SST; Technomedica Co., Tokyo, Japan) from the patients on the day of oocyte retrieval and centrifuged at 1630 x g for 30 min. Serum was stored at -20°C until use. The concentration of estradiol-17ß was assayed with an E2 kit, Daiichi II (Daiichi Radioisotope Laboratories, Tokyo, Japan). The intraassay and interassay coefficients of variation were 1.8–3.3% and 2.8–7.5%, respectively.

Quantification of Phosphatidylcholines (PCs), LPCs, and LPAs

Human follicular fluids were incubated at 37°C for 48 h; 0.5-ml aliquots of follicular fluid were withdrawn at 6, 12, 24, and 48 h and diluted with 2% KCl to 2 ml. Human serum was also incubated at 37°C for 48 h, and 0.3-ml aliquots were withdrawn at 12, 24, and 48 h. Volumes of 0.4 ml were withdrawn at 0 and 6 h for quantification of LPA. For determinations of PC and LPC, 0.2-ml samples were withdrawn at 0 and 6 h. These samples were diluted with 2% KCl to 2 ml. For quantification of PC, LPC, and LPA, 17:0 (hepatadecanoyl)/17:0 PC (160 µg/ml of sample), 17:0 LPC (70 µg/ml of sample), and 17:0 LPA (28 µg/ml of sample) were added as internal standards, respectively, to the diluted samples, and lipids were extracted by the method of Bligh and Dyer [21] after adjusting the pH of the diluted samples to below 2.5. Lipid extracts were then subjected to thin-layer chromatography (TLC) on Merck (Darmstadt, Germany) silica gel 60 plates developed with a solvent system of chloroform:methanol:20% ammonium hydroxide (60:35:8, v:v). Bands of PC, LPC, and LPA were scraped off the plates and suspended in 2 ml of water. PC and LPC were recovered from the silica gels by the method of Bligh and Dyer [21], and LPA was eluted from the silica gel suspensions acidified (pH 2.5) by the same extraction procedures. Conversions of PCs, LPCs, and LPAs to fatty acyl methyl esters were done in 0.5 ml 5% HCl in methanol at 100°C for 3 h, and the resultant methyl esters were extracted three times with n-hexane. They were quantified against heptadecanoic acid methyl esters derived from the internal standards (17:0/17:0 PC, 17:0 LPC, 17:0 LPA) in a Shimadzu (Kyoto, Japan) GC-15A gas chromatograph with a capillary column (J & W Scientific; DB-225, 30 m x 0.24-mm i.d., 0.25-µm thickness). The column temperature was kept at 120°C for 1 min and then increased to 220°C at 10°C/min. The temperature of the detector and injection ports was 250°C.

Measurement of LPLD Activity

LPLD activities in human follicular fluids and serum were measured essentially as described previously for those in rat plasma [12]. Briefly, body fluids (0.9 ml) were incubated with 0.1 volume of a solution of 14C-LPC (1-[palmityl-1-14C]-2-lyso-sn-glycero-3-phosphocholine, 2.035 Gbq/mmol; DuPont-NEN, Boston, MA) in saline containing 0.25% BSA (7 nmol/ml, 0.0154 MBq/ml) at 37°C for 6 h. At 0, 2, 4, and 6 h, 0.2-ml aliquots were withdrawn and diluted to 2 ml with 2% KCl. Lipids were extracted by the method of Bligh and Dyer [21] after acidification of samples (pH 2.5) and separated by TLC with chloroform:methanol:20% ammonium hydroxide (60:35:8) together with carrier LPA (0.1 µmol). The percentage conversions of 14C-LPC to LPA and PC were calculated from the radioactivities of bands on the TLC plates and expressed in terms of percentage per hour.

Statistical Analysis

Values are expressed as means ± SEM. Statistical analysis was carried out by Student's t-test. Values of p < 0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In ovarian follicles, nonvascularized granulosa cells are in direct contact with the follicular fluid but are separated from thecal blood capillaries by the basement membrane [14]. This membrane constitutes a blood-follicular barrier that acts as a molecular sieve allowing passage of proteins in inverse proportion to their molecular weights. There are reports of the predominance of high-density lipoprotein (HDL) and absence of low-density lipoprotein, very low density lipoprotein, and chylomicrons in human preovulatory follicular fluid [22, 23]. Consistent with these findings, the concentrations of PCs in human follicular fluids collected from women undergoing IVF were much less (251 ± 36 nmol/ml) than those in their serum (1136 ± 81 nmol/ml). The most abundant fatty acyl group attached to PCs in the follicular fluid was the palmitoyl group (16:0), followed by the stearoyl (18:0), linoleoyl (18:2), oleoyl (18:1), and arachidonyl groups (20:4) in decreasing order (Fig. 1, upper panel). PC in human serum had a fatty acid composition similar to that of PC in the follicular fluids except that 18:2 was more abundant than 18:0 (Fig. 2, upper panel). The concentrations of total LPC in the follicular fluids were 157 ± 24 nmol/ml, which were less than those in the serum (201 ± 12 nmol/ml), but the differences in the LPC concentrations in the two biological fluids were much smaller than those in the PC concentrations as described above. The fatty acid composition of LPC in fresh follicular fluid (16:0 > 18:0 > 18:2 = 18:1 > 20:4) was similar to that in fresh serum (Figs. 1 and 2, middle panels). The total amounts of LPA in the follicular fluids (25.3 ± 3.6 nmol/ml) were higher than in the serum (15.5 ± 2.2 nmol/ml). The most abundant LPA was 16:0 for both preparations, but the follicular fluids contained more unsaturated LPAs than the serum preparations, as shown in the lower panels of Figures 1 and 2.



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  		FIG. 1. Changes during incubation at 37°C in concentrations of PCs, LPCs, and LPAs in follicular fluids of women programmed for IVF. Values are means ± SEM for separate experiments on five patients. Symbols: 16:0 (open circles), 18:0 (solid circles), 18:1 (open triangles), 18:2 (solid triangles), and 20:4 (squares).



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  		FIG. 2. Changes during incubation at 37°C in concentrations of PCs, LPCs, and LPAs in serum of patients programmed for IVF. Symbols are as for Figure 1.

Lecithin-cholesterol acyltransferase (LCAT) activity in human follicular fluid was reported to be 17% of that in human plasma [24]. This would be relevant to a finding that only 10% of the total cholesterol is in a nonesterified form [25]. In accordance with previous results, the decreases in fatty acid concentrations of PC and increases in saturated LPCs in human follicular fluids after 6 and 12 h of incubation were much smaller than those in human serum (Figs. 1 and 2, upper panels). The persistent increases in amounts of saturated LPCs in the serum would mask their slow, persistent conversions to LPAs, whereas persistent decreases in polyunsaturated LPCs were obvious because there was no rapid formation of LPCs by LCAT activity (Figs. 1 and 2, middle and lower panels). The total amounts of LPA accumulated during 48-h incubation of follicular fluid samples from five women were 39.1 ± 6.2 nmol/ml, which is less than the 89.4 ± 29.2 nmol/ml for five serum samples from the same women. Table 1 shows the amounts of LPAs generated when three samples of follicular fluids from a single woman were incubated at 37°C for 24 h. Values are expressed as pmol/ml of follicular fluid/h. The most abundant species of LPA produced was 16:0, followed in decreasing order by 18:2, 18:1, 18:0, and 20:4. However, the order of their percentage conversions to LPCs on the basis of the amounts of LPCs in fresh follicular fluids was 20:4 > 18:2 > 18:1 > 16:0 > 18:0, indicating preference of LPLD for unsaturated LPCs over saturated LPCs.


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  		TABLE 1. Production of different LPAs by LPLD in incubated human follicular fluid.

Since the rate of LPA accumulation gradually decreased during 48-h incubation, we attempted to assess the initial rate of LPA accumulation by measuring the percentage conversion of radioactive 16:0 LPC added to the follicular fluid. Figure 3 shows typical results for these experiments. Radioactive LPC was found to be converted to LPA and PC by LPLD and LCAT, respectively, at constant rates within 6 h. As shown in Table 1, values for LPA generation in incubated follicular fluids from five women were similar when assayed with exogenous substrate, and variations in the values for follicular fluids from the same patients were very low.



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  		FIG. 3. Time courses of metabolic conversions of 14C-LPC to LPA and PC during incubation of human follicular fluid at 37°C. Values are means ± SEM for three samples of follicular fluid collected from one woman. All error bars are within symbols.

The concentration of estradiol-17ß in serum of the patients was 1.88 ± 0.224 ng/ml. When assayed with radioactive 16:0 LPC, these serum samples had 0.433 ± 0.043%/h LPLD activity (n = 5).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The proteins in follicular fluid have filtered through the blood-follicle barrier and do not contain low-density lipoprotein, very low density lipoprotein, or chylomicrons. In accordance with previous reports [22–24], we found that the concentration of PC in human follicular fluids collected from women undergoing IVF were much less than those in serum from the same women. LPC, a substrate of LPLD, was reported to constitute 18.4% of the total phospholipids in whole human follicular fluid [23], a percentage significantly higher than that in human plasma (6%). More recently, Lepage et al. [24] reported that the concentration of LPC in human follicular fluid is 286 µM, which is nearly identical to that in human plasma (252 µM). They also examined the distribution of radioactive 16:0 LPC in human follicular fluid, and found that 89% of the radioactivity was associated with the albumin fraction and the remainder with the HDL fraction. From results on the chromatographic behaviors of radioactive LPC of follicular fluid on a molecular sieve and a quaternary methylamine column, they concluded that about half the LPC was tightly bound to albumin while the rest was in a soluble free state or weakly associated with albumin. These results suggest that a large portion of LPC in the circulation could penetrate into the follicular fluid in a free soluble, albumin-bound, or HDL-associated form, reaching an equilibrated concentration almost the same as that in the circulation. The current study showed that follicular fluids collected from women programmed for IVF had LPLD activity that generated LPAs from endogenous LPCs, although the LPLD activity in their follicular fluid was less than that in their serum. The LPLD in the follicular fluid may have originated in the blood circulation, since it showed preference for unsaturated LPCs over saturated LPCs, like LPLD in rat plasma [11]. If this was so, a considerable proportion of LPLD in human blood could penetrate the blood-follicle barrier, indicating its soluble nature or its existence in an associate form with HDL, such as LCAT. However, the possibility that LPLD is released from granulosa cells in the follicular fluid cannot be completely excluded.

Liscovitch and Amsterdam [26] reported that GnRH activates phospholipase D in ovarian granulosa cells and that its stimulatory action on steroidogenesis could be mimicked by exogenous phospholipase D from Streptomyces chromofuscus. In ovarian steroidogenic cells, a signaling pathway involving adenylate cyclase is important in steroidogenesis. It has been postulated that the enhancement of gonadotropin receptor-coupled adenylate cyclase activity on isolated bovine luteal cells by bacterial phospholipase D is mediated by its direct metabolite, phosphatidic acid [27]. However, van Dijk et al. [28] recently reported that exogenous phospholipase D from S. chromofuscus generated LPA, stimulated accumulation of the GTP-bound form of Ras, and activated mitogen-activated protein kinase and DNA synthesis in fibroblasts. They suggested that the bacterial phospholipase D utilized preexisting LPC in the outer membrane leaflet to produce LPA on the cell surface, and that the LPA thus formed rapidly diffused laterally to become associated with its specific receptors. From these findings, it is tempting to speculate that LPLD in human follicular fluid can produce bioactive LPAs by attacking not only LPCs in the follicular fluid but also LPCs in the plasma membrane of granulosa cells and oocytes. This is an interesting subject for further study.

It has been suggested that the ovarian hyperstimulation syndrome increases risk of ovarian cancer [29]. Xu et al. [30] reported that LPA activates ovarian cancer cells and that the plasma levels of LPA of patients with ovarian cancer are significantly higher than those in healthy controls [31]. The present study showed that LPA is present in follicular fluid from women with ovarian hyperstimulation for IVF and that it is accumulated further by incubation with LPLD at 37°C. In addition, the LPLD activity of serum from healthy women was only about half that of women programmed for IVF when assayed with radioactive 16:0 LPC (unpublished results). These findings suggest the pathophysiological significance of enhanced production of LPA in the blood and blood-derived fluids of patients with ovarian diseases and women after ovarian hyperstimulation. Indeed, Xu et al. [32] reported that ascites from ovarian cancer patients contains ovarian cancer-activating factors identified as various molecular species of LPA. They suggested that LPAs with a polyunsaturated acyl chain such as linoleate, arachidonate, or docosahexaenoate accounted for the activities of the ovarian cancer-activating factor, since these were much more potent than oleoyl-, palmitoyl-, and stearoyl-LPAs. This finding is interesting, since the LPLD in follicular fluids was found to produce unsaturated LPA preferentially to saturated LPAs. Moreover, malignant effusions of patients with ovarian cancer were reported to contain more LPA-like activities than those of patients with other malignant cancers when assayed by a bioassay on neurite retraction [33]. Surprisingly, the level reached nearly 100 µM. Extracellular production or release of LPA-like activity has been postulated in other systems such as conditioned medium of preadipocytes after {alpha}2-adrenergic stimulation [34] and the aqueous humor of the rabbit eye after corneal injury [35], although its source and properties of formation are unknown. We detected LPLD activity in plasma of the rat [11, 12], in rabbit and mouse plasma, and in fetal calf serum (unpublished results). The widely distributed properties of mammalian blood LPLD have ultimate physiological significance through supplying bioactive LPA continuously to peripheral tissues such as the ovary.


   FOOTNOTES
 
1 This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan. Back

2 Correspondence: Akira Tokumura, Faculty of Pharmaceutical Sciences, The University of Tokushima, 1–78 Shomachi, Tokushima 770–8505, Japan. FAX: 81 88 633 7248; tokumura{at}ph.tokushima-u.ac.jp Back

Accepted: February 25, 1999.

Received: November 30, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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