1 Department of Obstetrics and Gynecology and 2 Central Research Institute, Hokkaido University School of Medicine, N15, W7, Kita-Ku, Sapporo 060-8638, Japan
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Abstract |
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Key words: human chorionic gonadotrophin /in-vitro fertilization/macrophage migration inhibitory factor/ovulation
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Introduction |
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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, 1966; David, 1966
). Moreover, MIF is known as a mediator of macrophage adherence, phagocytosis and tumoricidal activity (Weiser et al., 1991
). 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., 1992
; Bernhagen et al., 1993
; Nishihira et al., 1993
).
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., 1989). The crystallization of MIF was performed, which showed the preliminary three-dimensional protein structure (Suzuki et al., 1994
). MIF was originally observed in activated T cells (Weiser et al., 1989
); however, it has been reported to be expressed in other tissues, such as eye lens (Wistow et al., 1993
; Matsuda et al., 1996
) and cultured GC from human ovary (Wada et al., 1997
).
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.
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Materials and methods |
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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 2838 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 ASepharose (Pharmacia, Uppsala, Sweden) according to the manufacturer's protocol. The specificity of the MIF antibody was confirmed by Western blot analysis (Matsuda et al., 1996).
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., 1987) 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 citratephosphate 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 KruskalWallis test, where Scheffe's test was used for post hoc comparison. Data of follicular fluid MIF concentrations of natural cycles were analysed using the MannWhitney 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.
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Results |
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Discussion |
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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., 1993; Calandra et al., 1994
, 1995
; Bacher et al., 1996
). 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., 1993
). 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., 1996a
,b
), 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
(TNF-
) (Calandra et al., 1994
), which has been reported to induce prostaglandins as important mediators in the process of ovulation (Killick and Elstein, 1987
; Wang et al., 1992
). 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., 1993
), which was localized in the ovary with varied intensity in response to HCG treatment (Zackrisson et al., 1996
). For these reasons, MIF is considered to be a candidate as a mediator in the regulation of ovulation probably acting in concert with TNF-
, 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., 1975; Hedin et al., 1987
; Reich et al., 1991
). 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., 1996
). 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 MIFat least in partcontributes to the process of ovulation. Further investigation in this area is currently under way.
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Acknowledgments |
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Notes |
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References |
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Submitted on October 9, 1997; accepted on October 23, 1998.