Induction of macrophage migration inhibitory factor in human ovary by human chorionic gonadotrophin

Shin-ichiro Wada1,3, Takayuki Kudo1, Masataka Kudo1, Noriaki Sakuragi1, Hitoshi Hareyama1, Jun Nishihira2 and Seiichiro Fujimoto1

1 Department of Obstetrics and Gynecology and 2 Central Research Institute, Hokkaido University School of Medicine, N15, W7, Kita-Ku, Sapporo 060-8638, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The role of macrophage migration inhibitory factor (MIF) in human ovarian function remains obscure. The aim of this study was to investigate how MIF was related to ovulation by quantitative analysis of serum, follicular fluid and culture medium of granulosa cells obtained from in-vitro fertilization (IVF) and embryo transfer patients. Serum MIF concentrations in ovarian stimulation cycles for IVF-embryo transfer were higher at day 1 (median 92.6 ng/ml), which took place 35 h after human chorionic gonadotrophin (HCG) administration and just before the retrieval of oocytes, than those before day –6 (12.1 ng/ml), at day –5 to about day 0 (17.5 ng/ml) or at day 2 to about day 14 (8.2 ng/ml). MIF concentrations in the follicular fluid (113.4 ng/ml) obtained in ovarian stimulation cycles for IVF-embryo transfer were significantly higher than in serum (72.0 ng/ml) collected at the same time. MIF concentrations in the follicular fluid in natural cycles were higher in the ovulatory phase (51.6 ng/ml) than in the late follicular phase (13.8 ng/ml). MIF concentrations in the culture media of granulosa cells increased from 3.2 ng/ml to 7.2 ng/ml with HCG stimulation, and decreased from 2.4 ng/ml to 1.2 ng/ml when stimulation was withheld. These results indicate that HCG can induce the elevation of serum and follicular fluid MIF concentrations through the stimulation of ovarian cells, and that MIF is probably involved in the mechanism of ovulation.

Key words: human chorionic gonadotrophin /in-vitro fertilization/macrophage migration inhibitory factor/ovulation


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The immune system, as well as the endocrine system, are profoundly involved in the regulation of ovarian function. In particular, cytokines play an important role in the processes of follicular atresia, ovulation and luteolysis (Adashi, 1990Go; Vinatier et al., 1995Go; Machelon and Émilie, 1997Go). For example, interleukin-1ß (IL-1ß) is known to be increased in its gene expression in granulosa cell (GC) and interstitial cell culture just before ovulation (Hurwitz et al., 1991Go), to promote the ovulation rate in the ovarian perfusion model (Brännström et al., 1993Go), to modulate steroidogenesis in cultured GC (Fukuoka et al., 1992Go), and to be connected with other ovarian functions. Among the functions of the ovary, ovulation is one of the most dynamic phenomena. It is accompanied by inflammatory events and is probably characterized by the sequential release of cytokines from macrophages (Vinatier et al., 1995Go). However, the precise role of immunological and inflammatory events in ovulation remains unclear.

Macrophage migration inhibitory factor (MIF) is a cytokine that acts on concentrating macrophages at sites of infection and contributes to cell-mediated immunity (Bloom and Bennett, 1966Go; David, 1966Go). Moreover, MIF is known as a mediator of macrophage adherence, phagocytosis and tumoricidal activity (Weiser et al., 1991Go). Recently, various novel characteristics of MIF, such as its affinity for glutathione and its pituitary-derived hormone-like action, have been reported (Blocki et al., 1992Go; Bernhagen et al., 1993Go; Nishihira et al., 1993Go).

Human MIF cDNA was isolated from a T-cell line, and has a molecular mass of about 12 kDa with 115 amino acid residues (Weiser et al., 1989Go). The crystallization of MIF was performed, which showed the preliminary three-dimensional protein structure (Suzuki et al., 1994Go). MIF was originally observed in activated T cells (Weiser et al., 1989Go); however, it has been reported to be expressed in other tissues, such as eye lens (Wistow et al., 1993Go; Matsuda et al., 1996Go) and cultured GC from human ovary (Wada et al., 1997Go).

In this study, to investigate how MIF is related to ovulation, MIF concentrations were measured in serum and follicular fluid obtained from in-vitro fertilization (IVF) and embryo transfer patients. Furthermore, the effect of human chorionic gonadotrophin (HCG) on the secretion of MIF from GC culture was also investigated.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
IVF-embryo transfer method in ovarian stimulation cycles
Ovarian stimulation was performed using a long protocol with 900 µg/day of buserelin acetate (Suprecur; Hoechst Pharmaceutical & Chemicals K.K., Tokyo, Japan), 300 IU of follicle stimulating hormone (FSH) (Fertinom-P; Serono Japan K.K., Tokyo, Japan) or 150 IU of human menopausal gonadotrophin (HMG) (Pergogreen; Serono Japan K.K.) and 10 000 IU of HCG (HCG Mochida; Mochida Pharmaceutical Co. Ltd., Tokyo, Japan). HCG administration (day 0) was carried out when the largest follicles had developed to 16 mm in diameter and when serum oestradiol concentrations had reached more than 200 pg/ml per one mature follicle. Transvaginal ultrasound-guided oocyte retrieval was performed 35 h after HCG injection. To enhance luteal support following the oocyte retrieval, 125 mg of progesterone (Proluton Depot; Nihon Schering K.K., Tokyo, Japan) was administered on days 1 and 8, and 3000 IU of HCG on days 3, 6 and 9, respectively.

Blood sampling from IVF-embryo transfer patients in the four phases of the ovarian stimulation cycle
Forty-three blood samples were obtained from eight patients (age range 28–38 years) undergoing IVF-embryo transfer with ovarian stimulation. Samples were collected when HMG administration was started, and serial sampling continued until day 14. The cycle of each subject was divided into four phases: (i) before day –6; (ii) day –5 to about day 0; (iii) day 1 just before oocyte retrieval; and (iv) day 2 to about day 14. The samples were obtained between 9:00 and 13:00, and were preserved at –20°C for use in the assay of MIF, oestradiol, progesterone and HCG.

Blood and follicular fluid sampling at oocyte retrieval
Thirteen blood samples and 13 follicular fluid samples were obtained from 13 patients (aged 28 to ~39 years) undergoing IVF-embryo transfer with ovarian stimulation when oocyte retrievals were performed.

Follicular fluid sampling in the late follicular and ovulatory phases of the natural cycle
Nine follicular fluid samples in the late follicular phase were collected from nine patients (aged 33 to ~42 years) undergoing surgical operation in the natural cycle. Eight follicular fluid samples in the ovulatory phase were collected from eight patients (aged 30 to ~42 years) for IVF-embryo transfer, which was performed 35 h later as HCG was administered in the natural cycle. The samples were centrifuged at 200 g for 5 min to remove cellular components and stored at –20°C until assay of MIF.

GC cultures
Samples of follicular fluid from five patients for IVF-embryo transfer were centrifuged at 200 g for 5 min. The supernatant was decanted and cells were washed once with Ham's-F10 (Irvine Scientific, Santa Ana, CA, USA), layered on to 50% Percoll, and re-centrifuged at 200 g for 30 min to remove most of the red blood cells. Floating cells were aspirated from the interface, which included mainly GC and white blood cells, re-washed with Ham's-F10, and seeded in two wells at 2x104 cells per well in 1 ml Ham's-F10 with 10% fetal bovine serum. After the cells were cultured for 24 h in the wells to allow for preincubation and change of media, it was confirmed that the cells were attached on wells. After incubation of the GC for 24h, the media in two wells were changed (at D-1). HCG (0.5 IU/l) was included in the medium in one well, but excluded from the second well, which served as a control. After a further 24 h incubation, the media were removed and preserved (at D-2). All samples were centrifuged at 400 g for 30 min and the supernatants removed and stored at –20°C for use in assays of MIF, oestradiol and progesterone.

Preparation of anti-human MIF antibody
Polyclonal anti-human MIF serum was generated by immunizing New Zealand White rabbits with purified recombinant human MIF produced using the procedure described by Nishihira et al. (1993). The rabbits were inoculated intradermally with 100 µg of human MIF diluted in Freund's complete adjuvant (Wako, Osaka, Japan) at weeks 1 and 2, and with 50 µg of MIF diluted in Freund's incomplete adjuvant (Wako) at week 4. Immune serum was collected 1 week after the last inoculation. The immunoglobulin G (IgG) fraction (4 mg/ml) was prepared using Protein A–Sepharose (Pharmacia, Uppsala, Sweden) according to the manufacturer's protocol. The specificity of the MIF antibody was confirmed by Western blot analysis (Matsuda et al., 1996Go).

Assays of MIF
Specimens were assayed using an enzyme-linked immunosorbent assay (ELISA) method. Anti-human MIF antibody dissolved in 50 µl phosphate-buffered saline (PBS) pH 7.4 and diluted 1/1000 (4 µg/ml) was added to each well of a 96-well microtitre plate, which was then left for 1 h at room temperature. The plate was washed three times with PBS. All wells were filled with PBS containing 1% bovine serum albumin (Sigma Chemical Co., St Louis, MO, USA) for blocking and left for 1 h at room temperature. After removal of the blocking solution and washing three times with PBS containing 0.05% Tween 20 (washing buffer; Sigma), serum samples (50 µl) were added in duplicate to individual wells and incubated for 1 h at room temperature.

After washing the plate three times with washing buffer, 50 µl of biotin-conjugated (Gretch et al., 1987Go) anti-human MIF antibody was added. Following incubation for 1 h at room temperature, the plate was again washed three times with washing buffer, after which avidin-conjugated goat anti-rabbit IgG (Amersham International plc, Bucks, UK) was added to individual wells and incubated for 1 h at room temperature. The plate was again washed three times with washing buffer. 50 µl of substrate containing 200 µg of o-phenylethylenediamine (Wako) and 10 µl of 30% hydrogen peroxide in citrate–phosphate buffer (pH 5.0) adjusted with citric acid (0.05 M) and disodium hydrogen phosphate (0.1 M) was added to each well. After incubation for 20 min at room temperature, the reaction was stopped with 50 µl of 0.5 M sulphuric acid. The absorbance at 492 nm was measured with an ELISA plate reader (Bio-Rad, Model 3550).

Assays of oestradiol, progesterone and HCG
Assays of oestradiol, progesterone and HCG were performed using ELISA methods employing commercial kits, Serono SR-1 E2, Serono SR-1 P4 and Serono SR-1 hCG, respectively.

The intra-assay coefficients of variation (CV) for oestradiol were 9.1, 10.2 and 7.9% for pools of low, medium and high concentration, respectively; the corresponding inter-assay CV were 11.2, 9.3 and 10.5%. For progesterone, the corresponding intra- and inter-assay CV were 9.7, 5.3 and 11.4%, and 14.7, 7.8 and 7.0%. Similarly, for HCG, the intra- and inter-assay CV were 4.6, 3.5 and 3.5%, and 6.4, 4.2 and 7.4%, respectively.

Statistical analysis
The statistical significance of the data of serum MIF, oestradiol, progesterone and HCG concentrations at four phases in the ovarian stimulation cycle was determined using the Kruskal–Wallis test, where Scheffe's test was used for post hoc comparison. Data of follicular fluid MIF concentrations of natural cycles were analysed using the Mann–Whitney U-test. Data of comparison between serum and follicular fluid MIF concentrations, and of changes in MIF, oestradiol and progesterone in culture medium were analysed using the Wilcoxon signed-rank test.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Serum MIF, oestradiol, progesterone and HCG concentrations in ovarian stimulation cycles at four phases
Median (range) serum MIF concentrations in ovarian stimulation cycles were 12.1 (4.1–24.5) ng/ml before day –6, 17.5 (5.1–48.1) ng/ml at day –5 to ~0, 92.6 (29.3–122.5) ng/ml at day 1, and 8.2 (5.1–20.1) ng/ml at day 2 to ~14 (Figure 1Go). Serum MIF concentrations at day 1 were significantly higher (P < 0.001) than those of the other phases. Serum MIF concentrations were shown to be increased when those of both HCG and oestradiol were elevated.



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Figure 1. Box plots of serum macrophage migration inhibitory factor (MIF) concentrations at four phases in ovarian stimulation cycles. Day 0: when human chorionic gonadotrophin (HCG) was administered. Day 1: just before oocyte retrieval. Open circles, 10th–90th centiles; bars, 25th–75th centiles; boxes, median values. The value at day 1 is significantly (P < 0.001) higher than that in the other three phases.

 
MIF concentrations in serum and follicular fluid at oocyte retrieval in ovarian stimulation cycles
Median (range) MIF concentrations at oocyte retrieval were 72.0 (23.1–125.9) ng/ml in serum samples and 113.4 (32.4–251.3) ng/ml in follicular fluid samples (Figure 2Go). The difference was statistically significant (P < 0.01).



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Figure 2. Box plots of MIF concentrations in serum and follicular fluid at the time of oocyte retrieval in ovarian stimulation cycles. Open circles, 10th–90th centiles; bars, 25th–75th centiles; boxes, median values. Follicular fluid versus serum: P < 0.01.

 
Follicular fluid MIF concentrations in the late follicular and ovulatory phases in natural cycles
Median (range) concentrations of MIF in follicular fluid were 13.8 (6.9–31.9) ng/ml in the late follicular phase and 51.6 (29.8–143.9) ng/ml in the ovulatory phase (Figure 3Go). The difference was statistically significant (P < 0.01).



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Figure 3. Box plots of follicular fluid MIF concentrations in the natural cycle. Open circles, 10th–90th centiles; bars, 25th–75th centiles; boxes, median values. Late follicular phase versus ovulatory phase: P < 0.01.

 
MIF, oestradiol and progesterone concentrations in GC culture supernatants
With the addition 0.5 IU/l of HCG, median (range) concentrations of MIF in the supernatant of culture media were increased from 3.2 (2.8–4.1) ng/ml at D-1 to 7.2 (3.8–15.9) ng/ml at D-2 (P < 0.05). Without addition of HCG, MIF concentration fell from 2.4 (2.1–4.1) ng/ml at D-1 to 1.2 (1.0–3.2) ng/ml at D-2 (Figure 4Go). Median oestradiol concentrations changed from 2412 (1402–2987) ng/ml at D-1 to 2448 (489–5330) ng/ml at D-2 in HCG-containing media, and from 2098 (1826–3023) ng/ml at D-1 to 2482 (1441–2709) ng/ml at D-2 in HCG-free (control) media. Progesterone concentrations changed from 106 (32–135) ng/ml at D-1 to 253 (77–709) ng/ml at D-2 in HCG-containing media, and from 83 (76–120) ng/ml at D-1 to 66 (50–115) ng/ml at D-2 in HCG-free media. Progesterone concentrations in HCG-containing media between D-1 and D-2 were significantly different (P < 0.05), as were those at D-2 between HCG-containing and HCG-free media (P < 0.05).



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Figure 4. Time course of MIF concentrations in culture media, represented with box plots. Hatched column: HCG-containing medium. Open column: HCG-free medium. Bars indicate 10th–90th centiles; boxes indicate 25th–75th centiles and medians. D-1, 24 h incubation without HCG; D-2, the next 24 h incubation with or without HCG. Statistical analysis: a versus b, b versus c: P < 0.05.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Changes in serum MIF concentrations were measured in patients undergoing IVF-embryo transfer in ovarian stimulation cycles, showing that maximum values were reached at oocyte retrieval. Based on the data from the analysis of follicular fluid MIF concentration, HCG injection seems to have increased MIF concentrations in follicular fluid. Furthermore, the elevation of MIF concentrations by HCG stimulation was observed in the culture media from GC. The ovary is the main target organ of HCG, so these data strongly suggest that GC or the other cells in the ovary, such as infiltrating leukocytes, may produce MIF in response to HCG stimulation, thereby resulting in an increase in MIF concentrations in follicular fluid and the circulating blood. Ovulation is considered to be a type of inflammation, based on its rapid induction of immunological responses. In addition, the biosynthesis of MIF in cultured GC using immunohistochemistry, Western blot analysis and RT–PCR has previously been shown (Wada et al., 1997Go). Therefore, it can be postulated that MIF may be connected to some ovulatory mechanism.

In recent reports, it has been shown that MIF is released from macrophages, anterior pituitary cells and other cells in response to various inflammatory conditions or physiological stress (Bernhagen et al., 1993Go; Calandra et al., 1994Go, 1995Go; Bacher et al., 1996Go). The pituitary-derived MIF, secreted in response to stimulation of endotoxin, contributes to circulating MIF present in the post-acute phase of endotoxaemia (Bernhagen et al., 1993Go). This MIF response suggests that MIF circulates at low concentrations in the serum, but its release into the circulation is increased in response to a variety of stimulations. In the ovulatory phase, various inflammatory reactions are observed in the ovary. Therefore, it is speculated that circulating MIF is mainly secreted by ovarian cells in the ovulatory phase in addition to the basal secretion by macrophages, T cells, pituitary cells and other cells. Moreover, it was previously demonstrated that high concentrations of MIF were present in murine amniotic fluids and reproductive organs (Suzuki et al., 1996aGo,bGo), which revealed that MIF content varied according to the oestrus cycle. The mechanism of this change of MIF content remains unknown, although the fact demonstrates that MIF can be involved in ovarian functions. In addition, MIF is known to be expressed in response to tumour necrosis factor {alpha} (TNF-{alpha}) (Calandra et al., 1994Go), which has been reported to induce prostaglandins as important mediators in the process of ovulation (Killick and Elstein, 1987Go; Wang et al., 1992Go). On the other hand, MIF probably mediates its effects by way of other substances. It was reported that recombinant MIF induced nitric oxide (NO) synthase in murine macrophages (Cunha et al., 1993Go), which was localized in the ovary with varied intensity in response to HCG treatment (Zackrisson et al., 1996Go). For these reasons, MIF is considered to be a candidate as a mediator in the regulation of ovulation probably acting in concert with TNF-{alpha}, NO or other substances.

As for follicular rupture, luteinizing hormone (LH)-induced synthesis of prostaglandin endoperoxide synthase, plasminogen activators and collagenase by GC appears to be essential (Beers et al., 1975Go; Hedin et al., 1987Go; Reich et al., 1991Go). In particular, plasminogen can directly weaken the follicular wall and induce the activation of procollagenase to collagenase. It was reported that affinity purification of MIF/GIF (glycosylation inhibiting factor) in bovine brain was performed using a peptide ligand derived from a serpine that is homologous to plasminogen activator inhibitor-2, and to other serine proteases (Nishibori et al., 1996Go). These results suggest that MIF may contribute to weakening of the follicular wall in concert with plasminogen, though the precise mechanism remains unclear.

In conclusion, this study has demonstrated significant increases in MIF concentrations in serum and follicular fluid in response to HCG or LH stimulation. These present results indicate that GC and/or leukocytes in the ovary could be major sources of MIF in the ovulatory phase, which suggests that MIF–at least in part–contributes to the process of ovulation. Further investigation in this area is currently under way.


    Acknowledgments
 
We are grateful to Y.Mizue for assaying MIF, and to K.Yamaguchi for performing the hormone assays. This research was supported by a Grant-in-Aid for research (No. 07670162) from the Ministry of Education, Science and Culture of Japan, and by grants from the Akiyama and Ohyama Foundations.


    Notes
 
3 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Adashi, E.Y. (1990) The potential relevance of cytokines to ovarian physiology: the emerging role of resident ovarian cells of the white blood cell series. Endocr. Rev., 11, 454–464.[ISI][Medline]

Bacher, M., Metz, C.N., Calandra, T. et al. (1996) An essential regulatory role for macrophage migration inhibitory factor in T-cell activation. Proc. Natl. Acad. Sci. USA, 93, 7849–7854.[Abstract/Free Full Text]

Beers, W.H., Strickland, S. and Reich, E. (1975) Ovarian plasminogen activator: relationship to ovulation and hormonal regulation. Cell, 6, 387–394.[ISI][Medline]

Bernhagen, J., Calandra, T., Mitchell, R.A. et al. (1993) MIF is a pituitary-derived cytokine that potentiates lethal endotoxaemia. Nature, 365, 756–759.[ISI][Medline]

Blocki, F., Schilievert, P.M. and Wackett, L.P. (1992) Rat liver protein linking chemical and immunological detoxification systems. Nature, 360, 269–270.[ISI][Medline]

Bloom, B.R. and Bennett, B. (1966) Mechanism of a reaction in vitro associated with delayed-type hypersensitivity. Science, 153, 80.[ISI][Medline]

Brännström, M., Wang, L. and Norman, R.J. (1993) Ovulatory effect of interleukin-1ß on the perfused rat ovary. Endocrinology, 132, 399–404.[Abstract]

Calandra, T., Bernhagen, J., Mitchell, R.A. et al. (1994) The macrophage is an important and previously unrecognized source of macrophage migration inhibitory factor. J. Exp. Med., 179, 1895–1902.[Abstract]

Calandra T., Bernhagen J., Metz, C.N. et al. (1995) MIF as a glucocorticoid-induced modulator of cytokine production. Nature, 377, 68–71.[ISI][Medline]

Cunha, F.Q., Weiser, W.Y., David, J.R. et al. (1993) Recombinant migration inhibitory factor induces nitric oxide synthase in murine macrophages. J. Immunol., 150, 1908–1912.[Abstract/Free Full Text]

David, J.R. (1966) Delayed hypersensitivity in vitro: its mediation by cell-free substances formed by lymphoid cell-antigen interaction. Proc. Natl. Acad. Sci. USA, 56, 72–77.[ISI][Medline]

Fukuoka, M., Yasuda, K., Emi, N. et al. (1992) Cytokine modulation of progesterone and estradiol secretion in cultures of luteinized human granulosa cells. J. Clin. Endocrinol. Metab., 75, 254–258.[Abstract]

Gretch, D.R., Suter, M. and Stinski, M.F. (1987) The use of biotinylated monoclonal antibodies and streptavidin affinity chromatography to isolate herpesviruses hydrophobic proteins or glycoproteins. Anal. Biochem., 163, 270–277.[ISI][Medline]

Hedin, L., Gaddy-Kurten, D., Kurten, R. et al. (1987) Prostaglandin endoperoxide synthase in rat ovarian follicles: content, cellular distribution, and evidence for hormonal induction preceding ovulation. Endocrinology, 121, 722–731.[Abstract]

Hurwitz, A., Ricciarelli, E., Botero, L. et al. (1991) Endocrine- and autocrine-mediated regulation of rat ovarian (theca-interstitial) interleukin-1beta gene expression: gonadotropin-dependent preovulatory acquisition. Endocrinology, 129, 3427–3429.[Abstract]

Killick, S. and Elstein, M. (1987) Pharmacologic production of luteinized unruptured follicles by prostaglandin synthetase inhibitors. Fertil. Steril., 47, 773–777.[ISI][Medline]

Machelon, V. and Émilie, D. (1997) Production of ovarian cytokines and their role in ovulation in the mammalian ovary. Eur. Cytokine Netw., 8, 137–143.[ISI][Medline]

Matsuda, A., Tagawa, Y., Matsuda, H. et al. (1996) Identification and immunohistochemical localization of macrophage migration inhibitory factor in human cornea. FEBS Lett., 385, 225–228.[ISI][Medline]

Nishibori, M., Nakaya, N., Mori, S. et al. (1996) Affinity purification of macrophage migration inhibitory factor/glycosylation inhibitory factor (MIF/GIF) from bovine brain by using a peptide ligand derived from a novel serpine. Jpn. J. Pharmacol., 71, 259–262.[ISI][Medline]

Nishihira, J., Kuriyama, T., Nishino, H. et al. (1993) Purification and characterization of human macrophage migration inhibitory factor: evidence for specific binding to glutathione and formation of subunit structure. Biochem. Mol. Biol. Int., 31, 841.[ISI][Medline]

Reich, R., Daphna-Iken, D., Chun, S.Y. et al. (1991) Preovulatory changes in ovarian expression of collagenases and tissue metalloproteinase inhibitor messenger ribonucleic acid: role of eicosanoids. Endocrinology, 125, 1869–1875.

Suzuki, H., Nishihira, J., Koyama, Y. et al. (1996a) The role of macrophage migration inhibitory factor in pregnancy and development of murine embryos. Biochem. Mol. Biol. Int., 38, 409–416.[ISI][Medline]

Suzuki, H., Kanagawa, H. and Nishihira, J. (1996b) Evidence for the presence of macrophage migration inhibitory factor in murine reproductive organs and early embryos. Immunol. Lett., 51, 141–147.[ISI][Medline]

Suzuki, M., Murata, E., Tanaka., I. et al. (1994) Crystallization and a preliminary X-ray diffraction study of macrophage migration inhibitory factor from human lymphocytes. J. Mol. Biol., 235, 1141–1143.[ISI][Medline]

Vinatier, D., Dufour, P., Tordjeman-Rizzi, N. et al. (1995) Immunological aspects of ovarian function: role of the cytokines. Eur. J. Obstet. Gynecol. Reprod. Biol., 63, 155–168.[ISI][Medline]

Wada, S., Fujimoto, S., Mizue, Y. et al. (1997) Macrophage migration inhibitory factor in the human ovary: presence in the follicular fluids and production by granulosa cells. Biochem. Mol. Biol. Int., 41, 805–814.[ISI][Medline]

Wang, L.J., Brännström, M., Robertson, S.A. et al. (1992) Tumor necrosis factor {alpha} in the human ovary: presence in follicular fluid and effects on cell proliferation and prostaglandin production. Fertil. Steril., 58, 934–940.[ISI][Medline]

Weiser, W.Y., Temple, P.A., Witek-Giannotti, J.S. (1989) Molecular cloning of a cDNA encoding a human macrophage migration inhibitory factor. Proc. Natl Acad. Sci. USA, 86, 7522–7526.[Abstract]

Weiser, W.Y., Pozzi, L.M. and David, J.R. (1991) Human recombinant migration inhibitory factor activates human macrophages to kill Leishmania donovani. J. Immunol., 147, 2006–2011.[Abstract/Free Full Text]

Wistow, G.J., Shaughnessy, M.P., Lee, D.C. et al. (1993) A macrophage migration inhibitory factor is expressed in the differentiating cells of the eye lens. Proc. Natl. Acad. Sci. USA, 90, 1272–1275.[Abstract]

Zackrisson, U., Mikuni, M., Wallin, A. et al. (1996) Cell-specific localization of nitric oxide synthases (NOS) in the rat ovary during follicular development, ovulation and luteal formation. Hum. Reprod., 11, 2667–2673.[Abstract]

Submitted on October 9, 1997; accepted on October 23, 1998.