Gonadotropin-releasing hormone agonists induce meiotic maturation and degeneration of oocytes in the in vitro perfused rabbit ovary

Reproductive onimal.·.research~.: c·.c VoL 55, No.1, January 1991

FERTILITY AND STERILITY Copyright~

Printed on acid-free paper in U.S.A.

1991 The American Fertility Society

Gonadotropin-releasing hormone agonists induce meiotic maturation and degeneration of oocytes in the in vitro perfused rabbit ovary

Yasunori Yoshimura, M.D.* Yukio Nakamura, M.D. Haruhiko Yamada, M.D. Tomonori Nanno, M.D.

Yoshinobu Ubukata, M.D. Motomu Ando, M.D. Masahiko Suzuki, M.D.

Department of Obstetrics and Gynecology, Kyorin University School of Medicine, Tokyo, Japan

The present study was undertaken to assess the effects of gonadotropin-releasing hormone agonists (GnRH -a, buserelin and leuprolide acetate [LA]) on ovulation, oocyte maturation and degeneration, and steroid and prostaglandin production in the perfused rabbit ovary preparation. Ovulation did not occur in any of ovaries treated with buserelin or LA (10 2 to 104 ng/mL) in the absence of gonadotropin. Gonadotropin-releasing hormone agonists were associated with the resumption of meiosis in follicular oocytes in a dose-related manner. Furthermore, the addition of GnRH -a to the perfusate significantly increased the percentage of follicular oocytes that showed evidence of degeneration compared with contralateral untreated or human chorionic gonadotropin-treated controls. Prostaglandin E 2 and prostaglandin F 2.,. production by the perfused rabbit ovaries were stimulated significantly by GnRH -a treatment. Exposure to GnRH -a failed to increase either progesterone or estradiol production by the perfused rabbit ovaries. These data demonstrate that GnRH-a act directly in the rabbit ovary to trigger meiotic maturation in oocytes within the follicles, concomitantly increasing oocyte degeneration. Fertil Steril55:177, 1991

Gonadotropin-releasing hormone agonists (GnRH-a) have been used clinically in an attempt to reduce the incidence of premature spontaneous luteinizing hormone (LH) surges and to improve follicular stimulation in patients with prior failed cycles of in vitro fertilization (IVF) and embryo transfer. 1- 3 The addition of GnRH-a to human menopausal gonadotropin (hMG) has been shown to be beneficial in reducing the cycle cancellation rate, improving the oocyte recovery rate, and increasing the number of embryos available for transfer compared with cycles stimulated with hMG alone. 1 •3 •4 However, the direct action of GnRH -a on follicle-enclosed oocytes remains to be established in humans.

Received March 27, 1990; revised and accepted July 30, 1990. *Reprint requests: Yasunori Yoshimura, M.D., Department of Obstetrics and Gynecology, Kyorin University School of Medicine, 6-20-2, Shinkawa, Mitaka, Tokyo, Japan, 181. Vol. 55, No.1, January 1991

Gonadotropin-releasing hormone and its agonistic analogs exert direct inhibitory and stimulatory effects on ovarian function in mammals. 5 Stimulatory responses to GnRH -a, including ovulation and prostaglandin (PG) production, have been observed in hypophysectomized rats at proestrus in vivo 6 and in rat granulosa cells in vitro. 7 In addition, both GnRH and its agonists stimulate the meiotic maturation of follicle-enclosed rat oocytes in vitro. 8 - 10 However, evidence for direct effects of GnRH on the ovary in species other than the rat is either lacking or contradictory. 5 Whether GnRH action is species-specific remains to be determined. Perfusion of an isolated ovary provides an opportunity to conduct detailed studies of the intact organ under carefully regulated conditions independent of systemic inftuences. 11 The present study was undertaken to assess the effects of GnRH -a analogs on ovulation, oocyte maturation, and steroids and PG production in the perfused rabbit Yoshimura et al.

Effects of GnRH on rabbit ovary

177

ovary preparation. This report demonstrates the acute stimulatory effects of GnRH-a on ovarian function, including meiotic maturation of oocytes and PG production by preovulatory follicles. MATERIALS AND METHODS Animals

Thirty-nine sexually mature, female Japanese White rabbits, weighing 3.0 to 3.5 kg, were isolated for a minimum of 3 weeks and used in all experiments. The rabbits were housed individually under controlled lighting (14 hours of light, 10 hours of darkness) and temperature and fed water and Purina rabbit chow (Clea Japan Inc., Tokyo, Japan) ad libitum. Rabbits were anesthetized with intravenous 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

Each ovarian artery was cannulated in situ after all major vascular connections had been ligated. The ovary, with its artery, vein, and supporting adipose tissue still attached, was removed from the rabbit and immediately placed in a perfusion chamber. The perfusion fluid consisted of a total of 150 mL medium 199 (Gibco, Grand Island, NY) containing 1% bovine serum albumin (BSA; Sigma Chemical Co., St. Louis, MO), which was supplemented with heparin sulfate, insulin, streptomycin, and penicillin G, and adjusted to a pH of 7.4. The technique for cannulation and perfusion has been described in detail previously. 12 In a modification of this technique, 1% BSA was added to the basic perfusion fluid to increase oncotic pressure and decrease edema formation. Ovaries were observed periodically for evidence of follicle growth and 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 chorionic gonadotropin (hCG) or GnRH-a, 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 state of maturity and signs of degeneration. Oocytes were placed on a 178

Yoshimura et al.

Effects of GnRH on rabbit ovary

slide, fixed in 2.5% glutaraldehyde, and then stained with 0.25% lacmoid in 45% acetic acid for microscopic evaluation. The oocytes were classified on the basis of nuclear or chromosomal status and the presence of a first polar body. The degree of ovum maturity was expressed as the percentage of ova achieving germinal vesicle breakdown (GVBD). Ova were also assessed for degenerative changes including vacuolation, cytolysis, necrosis, fragmentation, and loss of spherical shape. Ovulatory efficiency, defined as the percentage of mature follicles that proceed to rupture, was calculated for each group. Experimental Design

The initial series of experiments (36 rabbits) was designed to determine whether GnRH-a were capable of inducing oocyte maturation and ovulation in rabbit ovaries in the absence of gonadotropin. Buserelin (D-Ser-[TBU] 6 -des-Gly-NH2 10 -LHRH ethylamide; Hoechst Japan, Tokyo, Japan) at a concentration of 102 , 103 , or 104 ng/mL, or leuprolide acetate (LA, D-Leu6 -[des-Gly10 -NH 2 ]-LHRH ethylamide acetate; Takeda Chemical Industries, LTD., Osaka, Japan) at a concentration of 102 , 103 , or 104 ng/mL was added to the perfusate of one ovary. The contralateral ovary of each rabbit was perfused simultaneously in a separate chamber with medium alone and served as a control. Six rabbits were used for each dose. Ovarian perfusion was carried out for 12 hours. The second experiment was designed to compare the ovulatory efficiency in hCG and GnRH-atreated ovaries. One ovary was perfused with 50 IU of hCG (CH-446, biological activity, 3,830 IU/mg; Organon, OSS, The Netherland) and served as a positive control. The contralateral ovary of each rabbit was perfused simultaneously with buserelin acetate 104 ng/mL or LA 104 ng/mL. Radioimmunoassay for Steroids and PGs

The concentrations of progesterone (P) and estradiol-17f1 (E 2 ) in the perfusate were measured by the direct, solid phase, 125 I -labeled steroid radioimmunoassay (RIA) kit manufactured by Diagnostic Products Corporation (Los Angeles, CA). Intra-assay and interassay coefficients of variation CVs were 7.2% and 7.9% for P and 5.3% and 6.4% for E 2 , respectively. The extraction of PGs was performed as described by Salmon and Flower 13 with minor modifications. After the addition of tritiated amounts Fertility and Sterility

Table 1

Follicular Oocyte Characteristics From Ovaries Perfused With Buserelin or LA Follicular oocytes No. ovaries perfused

No. ovaries ovulating

No.

GV"

GVBD

MI"

Mil"

18

0

93

89 (7)b

2 (1)

1 (0)

1 (0)

6 6 6 18

0 0 0 0

36 34 33 102

(7)

10 (2) 10 (3) 14 (5) 4 (2)

4 (2) 7 (2) 11 (2) 2 (0)

6 6 6

0 0 0

35 38 34

12 (5) 10 (3) 3 (1)

10 (2) 7 (1) 2 (0)

5 (1) 12 (2) 15 (5)

Control Buserelin (ng/mL) 102 103 104 Control LA(ng/mL) 102 103 104

"GV, germinal vesicle; MI, metaphase I; Mil, metaphase II.

of prostaglandin F 2, (PGF 2,) and prostaglandin E 2 (PGE 2 ) for the estimation of recovery, the culture medium samples (0.05 to 0.2 mL) were dissolved in 5 mL of ethyl acetone-methanol solution (2:1) to precipitate protein. The mixtures were centrifuged at 400 X g for 10 minutes, and 300 ~L of distilled water was added to the supernatants. Then, 4 mL of petroleum ether was added to the organic phase to separate unsubstituted fatty acids. The aqueous extract was acidified to pH 3 and extracted with ethyl acetate. The PG content of the organic phase was evaporated under a stream of nitrogen. The residues containing PGF 2, or PGE 2 were reconstituted in 500 ~L of phosphate buffer concentrate (0.05 M phosphate buffer, pH 7.3) containing 0.1% gelatin and 0.01% merthiolate. Prostaglandin E 2

.... .,. +I

...=..

100

V>

~ >-

...

= :e <.>

..... ....

':; ~ c: ~

....

~ Degeneration

•••

80

<.> 0 0

*** El GVBD

•••

E

••

60

•• 40

••

••

20

..m.. Control

10 2 10 3 10 1 Buserelin (ngjml)

leuprolide (ng;ml)

* P< 0.05, ** P
b

20 15 5 95

(3) (2) (1)

2 2 3 1

(1) (1) (1) (0)

8 (2) 9 (4) 14 (6)

Number of degenerated oocytes in parenthesis.

was converted to 11-deoxy-13,14 dihydro-15-ketoll{j,16-cyclo-PGE2 (bicyclic PGE 2 ) by incubation with 1 M sodium carbonate at 37oC for 24 hours. Aliquots of these extracts were used for RIA of PGs, and the determination of recovery rates of PGF 2, and bicyclic PGE 2 were 79.4% and 84.4%, respectively. The concentrations of PGF 2, and hicyclic PGE 2 were measured by RIA kits supplied by Amersham International Plc (Amersham, Bucks, United Kingdom). Prostaglandin F 2, and bicyclic PGE 2 antibodies showed <2% cross-reactivity with other closely-related PGs. The intra-assay and the interassay CVs were <15% and 20%, respectively. The sensitivities of the assays for PGF 2, and bicyclic PGE 2 were 3 pg/tube and 43 pgjtube, respectively. In both experiments, perfusate samples (2 mL) were withdrawn at the onset of perfusion and at 1, 2, 4, 6, 8, and 12 hours thereafter, and were replaced immediately by an equal volume of fresh medium with or without each GnRH -a. The samples were stored at -20oC until measurement of P, E 2 , PGF 2, , and PGE 2 by RIA. Statistical Analysis

All data regarding the time of ovulation, ovulatory efficiency, percent degeneration, percent GVBD, and the levels of P, E 2 , PGF 2, , and PGE 2 are presented as the mean ± SE. Statistical analysis was performed using Student's t-test. Differences in PG and steroid levels at each time point were compared by one-way analysis of variance.

RESULTS

In the first experiment, ovulation did not occur in any of the control ovaries or experimental ovaYoshimura et al.

Effects of GnRH on rabbit ovary

179

Table 2 Ovulation in Ovaries Perfused With HCG or With Buserelin or LA

HCG

Buserelin

50IU

No. ovaries perfused 12 No. ovaries ovulating 12 Ovulatory efficiency (%)" 81.8 ± 4.9b Time of ovulation 8.55 ± 0.37 Ovulated ova (n) 67 GVBDC(%) 89.6 ± 9.4 Degenerationd (%) 11.9 ± 2.1 Follicular oocyte (n) 15 GVBDC(%) 73.3 ±8.8 Degenerationd (%) 13.3 ± 1.2

Leuprolide acetate

Co')

...=

6 0

4

a:>

E

3

E ........._ bO

0

=

........ c.:J

0

2

c._

36 80.6 ± 8.6 30.5 ± 2.8e

34 85.3 ± 7.9 35.3 ± 3.1 e

ries treated with buserelin or LA at a concentration of 102 , 103 , or 104 ng/mL in the absence of gonadotropin (Table 1). The majority (95.7%) offollicular oocytes did not progress beyond the germinal vesicle stage in the control ovaries. GnRH-a, both buserelin and LA, led to the resumption of meiosis in the follicular oocytes in a dose-related manner (Fig. 1). The addition of buserelin (10 3 or 104 ng/ mL) or LA (10 2 , 103 , or 104 ng/mL) to the perfusate significantly increased the percentage of follicular oocytes, which showed evidence of degeneration compared with contralateral controls. In the second experiment, ovulation occurred in all ovaries treated with 50 IU ofhCG, whereas contralateral ovaries treated with buserelin 104 ng/mL or LA 104 ng/mL failed to ovulate (Table 2). The percentage of ovulated ova and follicular oocytes achieving GVBD in hCG-treated ovaries did not differ significantly from that of follicular oocytes in GnRH-a-treated ovaries. However, the exposure of perfused ovaries to each GnRH-a significantly increased the degeneration rate of follicular oocytes compared with the contralateral hCG-treated ovaries. Prostaglandin production by ovaries perfused with medium alone was very low throughout the entire perfusion period. The addition of each GnRH-a at 104 ng/mL to the perfusate significantly increased both PGE 2 and PGF 2" production after 4 and 6 hours of perfusion. The time course Yoshimura et al.

5

..... +I

a The percentage of mature follicles that proceeded to rupture. b Values are means ± SE. c The percentage of ovulated ova and follicular oocytes that achieved GVBD. d The percentage of ovulated ova and follicular oocytes that showed evidence of degeneration. e Significantly different from hCG-treated ovaries; P < 0.05.

180

0---0

o-o

10' ngfmL

6 0

control hCG (50 IU) ......... Buserelin (10 4ng/ml) •--..& leuprolide (10 4ng/ml)

6

Effects of GnRH on rabbit ovary

0

2

4

6

8

12

HOURS OF PERFUSION Figure 2 The production of PG E 2 by perfused rabbit ovaries. Rabbit ovaries were perfused with or without hCG (50 IU), buserelin (104 ng/mL), or LA (104 ng/mL) for 12 hours. Data represent the mean ± SE of six perfused rabbit ovaries.

of PG production in GnRH-a-treated ovaries was comparable with that in hCG-treated ovaries (Figs. 2 and 3). However, the amount of each prostanoid secreted into the perfusion media did not differ significantly for the different concentrations of GnRH-a (Fig. 4). Exposure to hCG significantly stimulated P synthesis by perfused rabbit ovaries within 1 hour, and

2.0

control hCG (50 IU) ......... Buserelin (10 4ng/ml) ·---· leuprolide ( 104ng/ml)

o-a

o-o

-.....

Co')

+I 1.5

...= a:>

E

1.0

0.5

0

2

4

6

8

12

HOURS OF PERFUSION Figure 3 The production of PGF2a by perfused rabbit ovaries. Rabbit ovaries were perfused with or without hCG {50IU), buserelin (104 ng/mL), or LA (104 ng/mL) for 12 hours. Data represent the mean ± SE of six perfused rabbit ovaries.

Fertility and Sterility

::::E .....

en

+I

..."'"' E

..."' E

"---

="'

..."'"'"'

.."'

Q...

Control

hCG 150 IU)

Buserelin (ng/ml)

Leuprolide (ng/ml)

Figure 4 The PGE 2 and PGF 2a concentrations in the perfusate of ovaries after administration of different concentrations of GnRH -a. Prostaglandin concentrations in the perfusate were determined after 12 hours of perfusion. Data represent the mean ± SE of six perfused ovaries.

shortly thereafter reached its maximum (Fig. 5). Estradiol production also was increased significantly in the perfusate of ovaries treated with hCG (Fig. 5). However, GnRH-a failed to increase both P and E 2 production by perfused rabbit ovaries. The levels of P and E 2 produced by GnRH -atreated ovaries did not differ significantly from those in control ovaries perfused with medium alone.

tropin-releasing hormone agonists at all dosages tested increased PGs synthesis by the perfused rabbit ovaries with similar stimulatory effects, whereas each GnRH -a induced meiotic maturation in a dose-related manner. We also found that resumption of meiosis of follicle-enclosed rabbit oocytes occurred in response to GnRH-a administration despite inhibition of PGs accumulation with indomethacin. 17 These data suggest that PG production stimulated by exposure to GnRH -a may not be obligatory for the resumption of meiosis. In another study using the same system, GnRH did not affect steroid production in the rabbit. 18 The present study also demonstrates that exposure to GnRH -a, at a dose inducing the resumption of meiosis, did not stimulate P and E 2 increases. Meiotic events can proceed in response to GnRH-a treatment in the absence of steroid production. The resumption of meiosis stimulated by GnRH-a may be induced by mechanisms other than through a rise in steroid concentrations. One cannot exclude the possibility, however, that the direct gonadal effects

..... en

300 control hCG (50 IU) ....... Buserelin (10 4ng/ml) ...... leuprolide ( 104ng/ml) o~

o--o

+I

..."'"' -=E 200

..."'

"---

DISCUSSION

The present study demonstrates that GnRH-a stimulated the meiotic maturation of follicular oocytes in the rabbits through direct effects on the ovary, but cannot induce ovulation in vitro in the absence of gonadotropin. These observations are contrary to the stimulatory effects of GnRH on ovulation observed in the in vitro perfused rat ovaries14 and hypophysectomized rats in vivo. 6·9 The local effects of GnRH on gonadal functions appear to be species-specific, since the availability of specific binding sites for GnRH in the gonad has been shown to vary between various species. 15·16 This species difference highlights the need for a re-evaluation of certain rodent models designed to investigate primate, and especially human, reproductive physiology. Gonadotropin-releasing hormone agonists stimulated PGs synthesis by perfused rabbit ovaries as well as the meiotic maturation of oocytes. GonadoVol. 55, No.1, January 1991

OJ~~~~~~~~ 0 1 2

12

1.0 ::::E ..... en

+I

..."'"'E E

..."'

0.5

"---

..

="'

-

....."'

0r-:-='F-'r----,----,---r----, 0 1 2 12 HOURS OF PERFUSION

Figure 5 The P and E 2 concentrations in the perfusion medium of rabbit ovaries. Rabbit ovaries were perfused with or without hCG (50 IU), buserelin (104 ng/mL), or LA (104 ng/ mL) for 12 hours. Data represent the mean± SE of six perfused ovaries. Yoshimura et al.

Effects of GnRH on rabbit ovary

181

of GnRH -a, including the stimulation of meiotic maturation and PGs synthesis, represent secondary pharmacological properties of this class of compounds. Exposure of rabbit ovaries to GnRH-a increased the degeneration rate of follicular oocytes as well as the stimulation of meiotic maturation. Ranta et al. 19 also have demonstrated that the most prominent morphological features of ovarian follicles in GnRH-a-treated rats were premature antrum formation with the degenerative changes in the granulosa cells and the degeneration and fragmentation of the oocytes. When ovarian follicle development was inhibited by multiple injections of GnRH -a, oocyte maturation was shown to be followed by a massive degeneration. 20 This degenerative phenomenon may be a long-term consequence for those oocytes that are stimulated by GnRH -a to resume meiosis while enclosed within the follicles. These findings indicate that pharmacological doses of GnRH and GnRH-a may exert a deleterious effect on ovum function, either by direct interaction with oocytes or indirectly through granulosa cell function. This calls for caution in the use of high doses of these analogs. Gonadotropin-releasing hormone or a GnRHlike material of ovarian origin has been showed to play a role as a physiological atretic signal in rats, since administration of a competitive antagonist increased the number of ovulable follicles. 21 Histological examination in human antral follicles revealed that the percentage of oocytes achieving G VBD in the follicles increased with the degree of follicular atresia. 22 These data suggest that both follicular atresia, including oocyte degeneration and resumption of meiosis, may be a closely correlated phenomena in GnRH -a treatment. In the present study using rabbits, however, GnRH-a did not cause oocyte degeneration in a dose-related manner. Banka and Erickson 22 investigated the possible relationship between follicular atresia and meiotic maturation induced by GnRH treatment in immature E 2-primed hypophysectomized rats. They found that early atresia in itself did not cause meiotic maturation, and the initiation of premature meiotic maturation by GnRH was specific for a subpopulation of tertiary follicles, those undergoing atresia. 23 The heterogeneity of oocyte response to GnRH -a appears to depend on differences in follicular development. In the human IVF program, the use of GnRH-a that eliminates functional endogenous gonadotropin production, has been associated with increased 182

Yoshimura et al.

Effects of GnRH on rabbit ovary

numbers of oocytes collected, fertilization rates, and pregnancy rates. 1- 4 Recent reports demonstrate, however, that concurrent initiation of GnRH -a and follicle-stimulating hormone causes a significant increase in oocyte atresia, resulting in a reduction in oocyte fertilization and embryo quality.24·25 The mechanisms governing follicular atresia remain to be clarified. The extremely high levels of LH generated early in the cycles may have stimulated an androgenic milieu conductive to atresia. In addition, the direct deleterious effects of GnRH -a on ovarian follicles may be responsible for follicular atresia, including oocyte degeneration. The recent finding that rat oocytes can respond to GnRH via direct interaction of the hormone with specific receptors 26 provides evidence that GnRH acts directly on the oocyte, inducing meiotic maturation and increasing oocyte degeneration. This raises questions regarding the potential use of GnRH agonistic analogs in fertility control. It is possible, however, that the laboratory rat and rabbit are not representatives of other species in respect to the direct gonadal effect of GnRH. In conclusion, we have demonstrated that GnRH-a are potent stimulators of the meiotic maturation of follicular oocytes in the rabbit ovaries. Gonadotropin-releasing hormone agonistic analogs may act directly in the rabbit ovary to trigger meiotic maturation in oocytes within the follicles while concomitantly increasing oocyte degeneration. This calls for caution in the use of high doses of these analogs for fertility control. REFERENCES 1. Neveu S, Hedon B, BringerJ, Chinchole J-M, Arnal F, Humeau C, Cristo! P, Viala J- L: Ovarian stimulation by a combination of a gonadotropin-releasing hormone agonist and gonadotropins for in vitro fertilization. Fertil Steril47:639, 1987 2. Caspi E, Ron-El R, Golan A, Nachum H, Herman A, Soffer Y, W einraub Z: Results of in vitro fertilization and embryo transfer by combined long-acting gonadotropin-releasing hormone analog D-Trp-6-luteinizing hormone-releasing hormone and gonadotropins. Fertil Steril51:95, 1989 3. V authier D, Lefebvre G: The use of gonadotropin-releasing hormone analogs for in vitro fertilization: comparison between the standard form and long-acting formulation of DTrp-6-luteinizing hormone releasing hormone. Fertil Steril 51:100, 1989 4. Palermo R, Amodeo G, Navot D, Rosenwaks Z, Cittadini E: Concomitant gonadotropin-releasing hormone agonist and menotropin treatment for the synchronized induction of multiple follicles. Fertil Steril 49:290, 1988 5. Knecht M, Ranta T, Feng P, Shinohara 0, Catt KJ: Gonadotropin-releasing hormone as a modulator of ovarian function. J Steroid Biochem 23:771, 1985

Fertility and Sterility

6. Ekholm C, Clark MR, Magnusson C, Isaksson 0, LeMaire W J: Ovulation induced by a gonadotropin releasing hormone analog in hypophysectomized rats involves prostaglandins. Endocrinology 110:288, 1982 7. Clark MR: Stimulation of progesterone and prostaglandin E accumulation by luteinizing hormone-releasing hormone (LHRH) and LHRH analogs in rat granulosa cells. Endocrinology 110:146, 1982 8. Hillensjii T, LeMaire W J: Gonadotropin releasing hormone agonists stimulate meiotic maturation of follicle-enclosed rat oocytes in vitro. Nature 287:145, 1980 9. Ekholm C, Hillensjii T, Isaksson 0: Gonadotropin releasing hormone agonists stimulate oocyte meiosis and ovulation in hypophysectomized rats. Endocrinology 108:2022, 1981 10. Dekel N, Sherizly I, Phillips DM, Nimrod A, Zilberstein M, Naor Z: Characterization of the maturational changes induced by a GnRH analogue in the rat ovarian follicle. J Reprod Fertil 75:461, 1985 11. Yoshimura Y, Wallach EE: Studies of the mechanism(s) of mammalian ovulation. Fertil Steril47:22, 1987 12. Lambertsen CJ, Jr, Greenbaum DF, Wright KH, Wallach EE: In vitro studies of ovulation in the perfused rabbit ovary. Fertil Steril27:178, 1976 13. Salmon JA, Flower RJ: Extraction and thin-layer chromatography of arachidonic acid metabolites. Methods Enzymol86:477, 1982 14. Koos RD, LeMaire WJ: The effects of a gonadotropin-releasing hormone agonist on ovulation and steroidogensis during perfusion of rabbit and rat ovaries in vitro. Endocrinology 116:628, 1985 15. Clayton RN, Huhtaniemi IT: Absence of gonadotropin-releasing hormone receptors in human gonadal tissue. Nature 299:56, 1982 16. Kiives K, Gottschall PE, Arimura A: Gonadotropin-releasing hormone binding sites in ovaries of normal cycling and persistent-estrus rats. Bioi Reprod 41:505, 1989

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17. Yoshimura Y: Unpublished data 18. Eisenberg E, Kitai H, Kobayashi Y, Santulli R, Wallach EE: Gonadotropin-releasing hormone: effects on the in vitro perfused rabbit ovary. Bioi Reprod 30:1216, 1984 19. Ranta T, Baukal A, Knecht M, Korhonen M, Catt KJ: Inhibitory action of a gonadotropin-releasing hormone agonist on ovarian follicle-stimulating hormone receptors and adenyl cyclase in vivo. Endocrinology 112:956, 1983 20. Naor Z, Zilberstein M, Zakut H, Linder H, Dekel N: Dissociation between the direct stimulatory and inhibitory effects of a gonadotropin releasing hormone analogue on ovarian function. Mol Cell Endocrinol31:261, 1983 21. Birnbaumer L, Shahabi N, Rivier J, Vale W: Evidence for a physiological role of gonadotropin-releasing hormone (GnRH) or GnRH-like material in the ovary. Endocrinology 116:1367, 1985 22. Banka CL, Erickson GF: Gonadotropin-releasing hormone induces classical meiotic maturation in subpopulations of atretic preantral follicles. Endocrinology 117:1500, 1985 23. Gougeon A, Testart J: Germinal vesicle breakdown in oocytes of human atretic follicles during the menstrual cycle. J Reprod Fertil 78:389, 1986 24. Brzyski RG, Muasher SJ, Droesch K, Simonetti S, Jones GS, Rosenwaks Z: Follicular atresia associated with concurrent initiation of gonadotropin-releasing hormone agonist and follicle-stimulating hormone for oocyte recruitment. Fertil Steril50:917, 1988 25. Loumaye E, Vankrieken L, Depreester S, Psalti I, de Cooman S, Thomas K: Hormonal changes induced by shortterm administration of a gonadotropin-releasing hormone agonist during ovarian hyperstimulation for in vitro fertilization and their consequences for embryo development. Fertil Steril51:105, 1989 26. Dekel N, Lewysohn 0, Ayalon D, Hazum E: Receptors for gonadotropin releasing hormone are present in rat oocytes. Endocrinology 123:1205, 1988

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