0013-7227/91/1291-0193$03.00/0 Endocrinology Copyright (p) 1991 by The Endocrine Society

Vol. 129, No. 1 Printed in U.S.A.

Involvement of Leukotriene B 4 in Ovulation in the Rabbit YASUNORI YOSHIMURA, YUKIO NAKAMURA, MAKOTO SHIRAKI, YUTAKA HIROTA, HARUHIKO YAMADA, MOTOMU ANDO, YOSHINOBU UBUKATA, AND MASAHIKO SUZUKI Department of Obstetrics and Gynecology, Kyorin University School of Medicine, Tokyo, Japan 181; Department of Obstetrics and Gynecology (M.S., Y.H.), Fujita-Gakuen Health University School of Medicine, Aichi, Japan

addition of NDGA at 10 5 M significantly inhibited hCG-stimulated LTB4 production by rabbit ovaries throughout the entire perfusion periods. The ovulatory efficiency in ovaries treated with hCG alone or with hCG plus NDGA correlated significantly with LTB4 production by perfused rabbit ovaries 6 h after exposure to hCG (« = 0.8893, P < 0.01). Furthermore, the addition of LTB4 at 100 ng/ml to the perfusate reversed the inhibitory effects of NDGA on hCG-induced ovulation. However, exposure to NDGA affected neither progesterone nor estradiol production elicited by hCG administration. These results suggest that NDGA may block hCG-induced ovulation in vitro, probably via the inhibition of LTB4 production by rabbit ovaries. (Endocrinology 129: 193-199, 1991)

ABSTRACT. The present study was undertaken to assess the effects of lipoxygenase products on ovulation, oocyte maturation, and steroid production in the perfused rabbit ovary preparation. Ovulatory efficiency was significantly reduced when rabbit ovaries were perfused with human CG (hCG) plus nordihydroguaiaretic acid (NDGA) at 10"5 or 10"6 M, as compared to contralateral hCG-treated controls. The addition of NDGA to the perfusate inhibited hCG-induced ovulation in a dose-related manner. The percentage of ovulated ova and follicular oocytes achieving germinal vesicle breakdown did not differ significantly between NDGA-treated ovaries and contralateral controls. Leukotriene B< (LTB4) production by the perfused rabbit ovaries reached its maximum 6 h after exposure to hCG and then declined. The

A

RACHIDONIC acid is metabolized by several enzyme systems. Cyclooxygenase leads to the formation of prostaglandins (PGs), including prostacyclin (PGI2), PGE2, PGF2a, and thromboxane. The lipoxygenase pathway leads to the formation of unstable hydroperoxyeicosatetraenoic acids and their stable breakdown products, hydroxyeicosatetraenoic acids (HETEs) and leukotrienes (1, 2). In the past decade, data have been reported suggesting that PGs may be involved locally in the process of ovulation in the ovary (3-6). In rabbits, the blockade of ovulation after exposure to a cyclooxygenatse inhibitor, indomethacin, can be reversed by the addition of PGF2« to the perfusate, further strengthening the idea of a role in ovulation (7). However, the recent studies raise questions about the specific role of PGs in the process of ovulation (8, 9). Data on the basis of the response to graded doses of indomethacin demonstrate no significant correlation between the ovulation rate and PG levels at the expected time of ovulation (8). This implies that there may be other indomethacin-sensitive mediators of ovulation which are produced independent

of PG biosynthesis. Such mediators may include histamine (10), kinins (11, 12), leukotrienes (13), and plasminogen activator (14), as well as other mediators of inflammations (15). The products of arachidonic acid metabolism formed via the lipoxygenase pathway, as well as the cyclooxygenase pathway, have been shown to play a role in the regulation of reproductive function (16-20). Since the intrabursal injection of specific inhibitors of lipoxygenase dose dependently reduced the number of ova released from treated ovaries (21), the lipoxygenase pathway may be involved in the process of follicle rupture. The in vitro rabbit ovarian perfusion system effectively isolates the ovary from systemic influences and permits the direct observation of ovulation under controlled experimental conditions (6, 11, 22). The present study was undertaken to assess the effects of lipoxygenase products on ovulation, oocyte maturation, and steroid production in the perfused rabbit ovary preparation. The report demonstrates the involvement of leukotriene B4 (LTB4) in the process of follicle rupture in the rabbit.

Received October 15,1990. Address all correspondence and requests for reprints to: Dr. Yasunori Yoshimura, Department of Obstetrics and Gynecology, Kyorin University School of Medicine, 6-20-2, Shinkawa, Mitaka, Tokyo, Japan 181.

Animals

Materials and Methods Sexually mature female Japanese White rabbits, weighing 3.0-3.5 kg, were isolated for a minimum of 3 weeks and used in

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194

LEUKOTRIENE B4 AND OVULATION

all experiments. The rabbits were housed individually under controlled lighting (14 h light, 10 h dark) and temperature and fed water and Purina rabbit chow (Clea Japan Inc., Tokyo, Japan) ad libitum. Rabbits were anesthetized iv with sodium pentobarbital (32 mg/kg), given heparin sulfate (120 U/kg) for anticoagulation, and then subjected to laparotomy. Ovaries were excluded from further study if they appeared immature or if 50% or more of the surface follicles appeared hemorrhagic. Ovarian perfusion Although the details of the perfusion system have been modified over the years, the basic components have remained constant (23). The ovarian artery and vein were separated for a distance of 2 cm from the ovary and were each dissected free from the surrounding adipose tissue, the peritoneum, and the adventitia. A fine glass cannula was inserted approximately 5 mm into the ovarian artery, and the vein was then cannulated using Teflon tubing (id, 0.01 in.; od, 0.028 in.) beveled at 45°. This cannula was inserted for a distance of 1 cm and secured in position. During this procedure, perfusate was steadily administered through the arterial cannula to confirm the free flow of fluid through the cannulated vein. Each ovary was then removed en bloc with the ovarian artery and vein and supportive adipose tissue, and immediately placed into a perfusion chamber. The perfusion medium (150 ml) was circulated at a rate of 1.5 ml/min. The basic perfusion fluid consisted of tissue culture medium 199 (GIBCO, Grand Island, NY) containing 1% BSA (Sigma Chemical Co., St. Louis, MO), which was supplemented with heparin sulfate (200 U/liter), insulin (20 U/ liter), streptomycin (50 mg/liter), and penicillin G (75 mg/ liter), and adjusted to a pH of 7.4. Ovaries were observed every 15 min for evidence of follicle rupture. The ovulated ovum surrounded by its cumulus mass was recovered carefully from the ovarian surface at the time of follicle rupture. Twelve hours after exposure to human CG (hCG), oocytes were recovered by aspiration from mature follicles (>1.5 mm in diameter), and the experiment was concluded. Both ovulated ova and follicular oocytes were assessed for the state of maturity and signs of degeneration. Cumulus cells were removed from oocytes by gentle agitation using a narrow-bore pipette, in a solution of hyaluronidase 0.5 mg/ml (Sigma Chemical Co.). Oocytes were placed on slides, fixed in 2.5% glutaraldehyde, and then stained with 0.25% lacmoid in 45% acetic acid for microscopic evaluation. Oocytes were classified on the basis of nuclear and chromosomal status and the presence of a first polar body. Dictyate oocytes were identified by the presence of an intact nucleus, germinal vesicle (GV). Oocytes in which the nuclear envelope had disappeared, and the chromosomes condensed were classified as GV breakdown (GVBD). Oocytes at metaphase I contained chromosomes aligned on a metaphase spindle, but no polar body. Oocytes with a first polar body were classified as metaphase II. The degree of oocyte maturity was expressed as the percentage of oocytes which had achieved GV breakdown (GVBD). Oocytes also were assessed for degenerative changes, including vacuolation, cytoplasmic flocculation, necrosis, fragmentation, karyorrhexis, and disintegration. Ovulatory efficiency, defined as the percentage of mature follicles which proceeded to rupture, was calculated for each group.

Endo«1991 Voll29«Nol

Experimental design The preliminary experiment using six rabbits was undertaken to determine if the addition of hCG to the perfusate stimulates leukotriene B4 production by the rabbit ovaries. Both ovaries were perfused with medium alone. Thirty minutes after the onset of perfusion, 50 IU hCG (CH-446, OSS, The Netherlands; biological activity, 3830 IU/mg) was added to the perfusate of one ovary. In the subsequent experiment, one ovary was perfused with medium alone and served as a control. The contralateral ovary of each rabbit was perfused simultaneously with nordihydroguaiaretic acid (NDGA, Sigma Chemical Co.) at a concentration of 10~5, 10"6, or 10~7 M in a separate chamber. Thirty minutes after the onset of perfusion, 50 IU hCG was added to the perfusate of both ovaries. Six rabbits were used for each dose of NDGA. Ovarian perfusion was carried out for 12 h after hCG exposure. Both arterial and venous samples were obtained at the start of perfusion and 0.5,1,2, 4, 6,8, and 12 h thereafter. Samples were stored at -70 C until progesterone, estradiol17/5, and LTB4 concentrations were determined. The final experiment was undertaken to assess if the inclusion of LTB4 in the perfusate reverses the reduced ovulatory efficiency in NDGA-treated ovaries. One ovary was perfused with NDGA at 10"5 M and served as a control. The contralateral ovary of each rabbit was perfused with LTB4 at 100 ng/ml plus NDGA at 10"5 M. Thirty minutes after the onset of perfusion, 50 IU hCG was added to the perfusate of both ovaries. Ovarian secretion rates Assays were performed on samples of the perfusate taken from the apparatus at a site just before perfusate entered the cannulated ovarian artery and also on samples of ovarian vein effluent at each time point specified above (24). The secretion rates of the hormones (Sh) were calculated as follows: Sh (ng/min) = [Vh (ng/ml) - Ah (ng/ml)] X flow rate (ml/min), where Vh = hormone concentration in ovarian venous effluent, and Ah = hormone concentration in the perfusate immediately before it enters the ovary. RIA for steroid and LTB4 The concentrations of progesterone (P) and estradiol-17/3 (E2) in the perfusate were measured by the direct, solid phase, 125 I-labeled steroid RIA kit manufactured by Diagnostic Products Corporation (DPC, Los Angeles, CA). Intraassay and Interassay coefficients of variation were 7.2% and 7.9%, respectively, for P, and 5.3% and 6.4%, respectively, for E2. The extraction of LTB4 was performed as described by Powell (25) with minor modifications. After the addition of tritiated LTB4 to estimate the recovery rate, the perfusate samples (0.2-0.5 ml) were mixed in 2 ml ice-cold ethyl acetatemethanol solution (2:1) to precipitate protein. The mixtures were centrifuged at 400 X g for 10 min at 4 C, and the aqueousacetone was then adjusted to pH 10-11 with 1 M sodium hydroxide. Two milliliters of petroleum ether were added to the organic phases to separate unsubstituted fatty acids. The aqueous extract was further extracted with 3 ml 0.05 N acetate buffer at pH 4.0, which served as the sample solution for application to the column. Solid phase extraction techniques

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LEUKOTRIENE B4 AND OVULATION were performed using a Bond Elut LRC C8 reversed phase column (100 mg, Analytichem International, Habor, CA). Before applying the sample, precondition of the columns with 5 ml methanol was followed by 5 ml distilled water. The samples were applied to the column and eluted in turn with 5 ml water, 5 ml hexane, and 5 ml hexane-ethyl acetate (95:5) to remove interfering compounds. The samples were then eluted with 5 ml hexane-ethyl acetate-ethanol (30:65:5). This fraction was taken to dryness and reconstituted in 500 n\ 50 mM tris-HCl buffer containing 0.1% gelatin. Aliquots of these solutions were used for the RIA of LTB4 and determinations of recovery rates. The average recovery rate was 78.9 ± 4.1% (mean ± SEM). The concentration of LTB4 was measured by the LTB4 [3H] assay reagents system supplied by Amersham International Pic. (Amersham, United Kingdom). LTB4 antibody cross-reacts less than 1% with other closely related leukotrienes (20-OH-LTB4 0.4%, 6-trans LTB4 0.4%, LTC4

Involvement of leukotriene B4 in ovulation in the rabbit.

The present study was undertaken to assess the effects of lipoxygenase products on ovulation, oocyte maturation, and steroid production in the perfuse...
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