1 Department of Pharmacology and 2 Laboratory of Biological Chemistry, University of Ioannina Medical School, Greece, 3 Protein & Peptide Group, EMBL, Heidelberg, Germany and 4 Department of Obstetrics and Gynaecology, University of Thessalia Medical School, 22 Papakiriazi Str., 412 22 Larissa, Greece
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Abstract |
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Key words: gonadotrophin surge attenuating factor/human follicular fluid/luteinizing hormone
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Introduction |
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GnSAF reduces only the GnRH-induced secretion of LH without affecting basal gonadotrophin secretion. It is a non-steroidal factor different from inhibin (Fowler et al., 1990). The latter is a gonadal protein involved in the control of FSH secretion from the pituitary (Ying, 1988
). Non-steroidal gonadal factors involved in the regulation of pituitary LH secretion have not yet been identified. It is likely that GnSAF is a new ovarian hormone that may play a role in the control of LH secretion during the mid-cycle LH surge in the female (Fowler and Templeton, 1996
).
By monitoring the change in the response of LH to GnRH in various in-vitro bioassay systems GnSAF/IF bioactivity has been demonstrated in ovarian follicular fluid of various species such as pigs (Danforth et al., 1987), rats (Busbridge et al., 1988
), monkeys (Schenken and Hodgen, 1986
), cows (Danforth and Cheng, 1994
) and humans (Busbridge et al., 1990
; Fowler et al., 1990
; Knight et al., 1990
; Mroueh et al., 1996
). During the last few years two publications have reported the purification of GnSAF/IF bioactivity to homogeneity. A 37 kDa protein from rat Sertoli cell-conditioned medium was isolated (Tio et al., 1994
) while a 69 kDa protein with GnSAF/IF activity from bovine follicular fluid has been purified (Danforth and Cheng, 1995
). So far, purification of GnSAF to homogeneity from human follicular fluid (FF) has not been reported.
In the present study we report the isolation, purification, and amino acid sequence of a polypeptide with GnSAF activity from FF.
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Materials and methods |
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GnSAF and inhibin bioassays
At the end of the plating period the cells' medium was replaced with SFDM and the cells were incubated in triplicate (on at least two separate cultures) in the absence (control) or presence of test substances for 48 h. Media were collected and stored at 20°C (basal secretion). Inhibin bioactivity is defined as inhibition of basal FSH secretion over this 48 h period. To assay for GnSAF the cells were washed with fresh medium and then incubated with 0.1 µmol/l GnRH plus test substances and they were incubated for an additional period of 4 h. After incubation, media were removed and frozen at 20°C (GnRH-induced LH secretion) until measurement for the levels of LH and FSH by enzyme linked immunosorbent assay (ELISA) methods. GnSAF activity is defined as the suppression of GnRH-stimulated LH secretion over this 4 h interval. One unit of GnSAF is the amount required to inhibit 50% of GnRH-induced LH secretion.
ELISA assays for the measurement of rat gonadotrophins
Rat gonadotrophins in cell culture media were measured in a competitive ELISA format, using reagents supplied by the NIDDK, as has been described (Pappa et al., 1999). Briefly, rat LH ELISA used hormone preparations NIDDK-rLH-RP3 and rLH-I-9 and antisera NIDDK-anti-rLH-S-11. Rat FSH ELISA used hormone preparations NIDDK-rFSH-RP3 and NIDDK-rFSH-I-8 and antisera NIDDK-anti-rFSH-S-11. The range of the assay for rat LH ELISA and rat FSH ELISA was 0.550 ng/ml and 1.2540 ng/ml respectively. In both ELISA methods the optical density decreases as a linear function of the LH or FSH concentration. The intra- and interassay coefficients of variation of sample pools containing high, medium and low LH concentrations were 8.8, 5.1 and 3.8 respectively in rat LH ELISA. The intra- and interassay coefficient of variation of the same pools containing high, medium and low FSH concentrations were 13.6, 6.9 and 7.7% respectively.
Heat treatment after dextran-coated charcoal
Human follicular fluid (FF) was obtained from superovulated women who had participated in an in-vitro fertilization programme in the Department of Obstetrics and Gynaecology, University of Ioannina, Greece. Informed consent was obtained from the women and the study was approved by the scientific committee of the hospital. Approximately 250 ml of frozen FF were thawed, centrifuged to remove precipitated proteins, and subjected to two-fold steroid extraction treatment as previously described (Danforth et al., 1987). After steroid extraction, FF was divided in 25 ml aliquots and heated in sealed flasks for 5 minutes at 80°C and centrifuged.
Heparin-sepharose chromatography
The heat treated FF was applied to five 1x20 cm heparin-sepharose (Pharmacia Biotech, Vienna, Austria) columns at 8 ml/h. Non-bound proteins were eluted with 20 mmol/l Tris-HCl buffer, pH 7.0 at 25°C and collected as a pool. The bound proteins were eluted at 8 ml/h with 20 mmol/l Tris-HCl buffer, pH 7.0 containing 1 mol/l NaCl. The pooled non-bound fractions from heparin-sepharose column were brought to a volume of 30 ml using an Amicon concentrator (UM2, Amicon Ltd, Stonehouse, Glos., UK). Aliquots of the pooled bound and non-bound fractions from the column were tested for GnSAF and inhibin activity.
Con A sepharose chromatography
The unbound fractions from heparin-sepharose chromatography containing GnSAF activity were applied to three 1x10 cm Con A sepharose chromatography (Pharmacia Biotech) columns developed in 20 mmol/l Tris-HCl/0.5 mol/l NaCl at 18 ml/h. After the columns were washed, the absorbed proteins were eluted by a linear gradient of 0.000.25 mol/l -D-methylmannoside in starting buffer. Individual fractions (4 ml each) were collected and equilibrated against SFDM medium using Centricon-3 microconcentrators (Amicon) prior to testing for GnSAF and inhibin bioactivity.
Vydac C4 reversed-phase high-performance liquid chromatography (HPLC)
The biologically active fractions obtained from the above step were pooled, concentrated and lyophilized, then resuspended in 5:1 solvent A/solvent B (solvent A: 0.1% TFA/H2O, solvent B: 0.1% TFA/CH3CN) at a final volume of 50 µl and loaded onto a Vydac reversed-phase HPLC column (4.6x250 mm internal diameter) at a flow rate of 1 ml/min. Bound fractions were eluted using a linear gradient of 2375% solvent B over a period of 45 min. Fractions of 1 ml were collected. Aliquots of these fractions were lyophilized and resuspended in culture medium prior to GnSAF and inhibin bioassays.
Preparative native rod gel electrophoresis
Fractions with maximum GnSAF activity after RP-HPLC were pooled and separated by native polyacrylamide gel electrophoresis (PAGE) onto a 7% rod polyacrylamide gel (inner diameter of gel tube: 0.5 cm, running conditions: 2 mA, 1 W max power). Following electrophoresis the rod gel was rinsed in PBS. Then, it was cut in 0.25 cm slices and each slice was halved. One half was used for the detection of GnSAF activity [SFDM culture medium (1 ml) was added to the gel pieces and after allowing the proteins to diffuse overnight at 4°C, the products of diffusion were tested in the bioassay]. The other half was used for protein analysis by sodium dodecyl sulphate (SDS)PAGE.
SDSPAGE and microsequencing
The halves of each 0.25 cm gel piece kept for analysis by SDSPAGE were immersed in 30 µl sample buffer (0.0625 mol/l Tris/HCl pH 6.8, 2.3% SDS, 0.025 mol/l dithiothreitol, 10% glycerol, 0.05% bromophenol blue) for 30 min at room temperature and analysed under reducing conditions in 1 mm thick 15% acrylamide gel, according to established methods (Laemmli, 1970). The proteins were revealed by silver staining. The following molecular weight standards were used to calibrate the gel: bovine albumin (Mr 66 000), egg albumin (Mr 45 000), glyceraldheyde-3-phosphate dehydrogenase (Mr 36 000), carbonic anhydrase (Mr 29 000), trypsinogen (Mr 24 000), trypsin inhibitor (Mr 20 100),
-lactalbumin (Mr 14 200). The GnSAF protein band was excised from the gel and was immersed in 1% acetic acid. It was then sent to EMBL (Heidelberg, Germany) for sequence analysis by the method of mass spectrometry as previously described (Shevchenko et al., 1996
).
Incubation with N-glycosidase F
Partially purified GnSAF (2.5 µg: the active fractions after the HPLC step) was incubated with N-glycosidase F in 50 mmol/l sodium phosphate buffer in the presence of 10 mmol/l EDTA, 50 mmol/l HEPES, 0.035% ß-mercaptoethanol, 0.5% NP-40, pH 7.5, in a final volume of 100 µl, at 37°C for 36 h. Ten units of N-glycosidase F were added at 0, 12 and 24 h in order to obtain extensive deglycosylation of proteins. Rabbit IgG (~1 µg) was used as control as it was subsequently subjected to SDSPAGE. The treated GnSAF fractions were tested for activity in the rat pituitary bioassay.
Statistical analysis
Analysis of variance, followed by Dunnett's or Bonferroni tests, was used to analyse all purification and dose response experiments. Significance was set at P < 0.05.
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Results |
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Discussion |
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GnSAF was found to have sequence homology to the 12.5 kDa C-terminal fragment of human serum albumin. If an N-terminus at amino acid 490 of HSA is assumed, the fragment with GnSAF activity would include the last loop of the nine loop-link-loop structures of HSA (He and Carter, 1992; Carter and Ho, 1994
). HSA belongs to a multigene family of proteins that includes
-fetoprotein and vitamin D-binding protein. It is the most multifunctional transport protein known to date (Carter and Ho, 1994
). The possibility of participation of the carboxy terminal fragment of HSA molecule to the regulation of LH secretion becomes a very interesting hypothesis. Of interest is that intact HSA molecule does not reduce GnRH-induced LH secretion (data not shown). Therefore, it could be assumed that the proteolytic cleavage of the molecule releases a specific regulatory activity. If the cleavage is not due to the purification conditions (something quite unlikely given the high stability of the albumin molecule), then it may become important to elucidate the role of specific proteases in follicular fluid under conditions of induced superovulation. However, the case of specific protein fragments exerting different biological activity from their intact proteins is not unknown. Endostatin and angiostatin are two such examples. Endostatin (mol. wt: 20 kDa) is a C-terminal fragment of collagen XVII (O'Reilly et al., 1997
), while angiostatin (mol. wt: 38 kDa) was identified as an internal fragment of plasminogen (O'Reilly et al., 1994
). Both of these proteins are inhibitors of angiogenesis, currently used against cancer, while the molecules they derive from are not.
As demonstrated in this report, GnSAF is possibly a glycoprotein, based on the observation that GnSAF activity was eluted in the bound fractions of Con A sepharose chromatography. Deglycosylation at the extent accomplished under the described conditions had no effect on the biological activity of GnSAF (Figure 4).
The present study describes for the first time the isolation of GnSAF from human follicular fluid. The estimated ED50 value for the final purification step (~30 ng) strongly indicates a regulatory role on LH secretion for this peptide, which is of physiological importance. The estimated molecular weight of 12.5 kDa protein is similar to that initially suspected for human GnSAF (Fowler et al., 1992) and different from that reported for GnSAF/IF isolated from Sertoli cell-conditioned medium (Tio et al., 1994
) and from bovine follicular fluid (Danforth and Cheng, 1995
). GnSAF bioactivity in the 1030 kDa size range has been demonstrated using crude serial ultrafiltration experiments (Fowler et al., 1992
), However, FF GnSAF in that study was not purified to homogeneity. A 37 kDa protein with GnSAF/IF activity from 32 l rat Sertoli cell-conditioned medium has been isolated (Tio et al., 1994
). Soon after this publication (Danforth and Cheng, 1995
) the purification of a 69 kDa protein with GnSAF activity from bovine follicular fluid was reported. Both these proteins are monomeric but it is doubtful if they bear any common structural similarities, as different NH2-terminal sequences were detected. Compared to these putative GnSAF proteins, GnSAF isolated from FF shows properties similar to both of them. Both GnSAF obtained from human and from bovine follicular fluid GnSAF suppress GnRH-induced LH secretion without affecting basal FSH secretion. It is interesting that in both purification procedures GnSAF activity and inhibin activity are separated at initial stages of purification. Conversely, GnSAF/IF isolated from Sertoli cell-conditioned medium demonstrates potent inhibin-like activity and causes reduction in both GnRH-induced LH secretion and basal FSH secretion. On the other hand, both human follicular fluid GnSAF and GnSAF isolated from rat Sertoli cell-conditioned medium proved to be resistant to treatment with acetonitrile, the organic solvent used in reverse phase HPLC. A highly purified preparation with GnSAF/IF activity from FF was described recently (Mroueh et al., 1996
), and was compared with GnSIF activity present in porcine follicular fluid. Human GnSAF caused reduction of GnRH-induced LH secretion and had no effect on basal FSH secretion. It was found that porcine GnSIF and human GnSAF have the same bioactivity in vitro and chromatographic characteristics. Antibodies raised against porcine GnSIF recognized two proteins with molecular weights of ~63 and ~59 kDa respectively (Mroueh et al., 1996
).
The discrepancies mentioned above raise questions about the number of existing proteins that demonstrate GnSAF activity. There might be more than one protein involved in the regulation of LH secretion from pituitary, exerting suppression of GnRH-induced LH secretion.
The assumption of the existence of a non-steroidal ovarian factor which regulates the LH surge was introduced in order to explain the clinical observations of unsuccessful ovulation due to reduced mid-cycle LH surge in women participating in in-vitro fertilization programmes (Messinis and Templeton, 1989, 1990a
,Messinis and Templeton, b
, 1991
; Messinis et al., 1991
). On the other hand, the existing information on the modulation of LH surge is inadequate to fully explain the regulation of the precise timing and the exact amplitude of the LH surge, which is required for the follicular maturation and ovulation. However, recent evidence has further clarified the role of GnSAF in the control of the amplitude of the mid-cycle LH surge as a factor that antagonizes the sensitizing effect of oestradiol on the pituitary (Messinis et al., 1998
). Nevertheless, the accumulating evidence on the regulation and existence of GnSAF/IF activity is very confusing. Although the production of GnSAF is considered to be stimulated by FSH in both rats and humans (Koppenaal et al., 1991
; Messinis et al., 1993
), recent results suggest that in the rat ovarian cycle GnSAF is not regulated by FSH (Tio et al., 1998
). Our present data also do not support the aspect that inhibin and GnSAF have mutual intrinsic bioactivities (Tio et al., 1994
). Full sequencing of human GnSAF has not yet been reported, and this is also true for the other isolated proteins with GnSAF activity. The sequence of these peptides will show if novel proteins that regulate human ovulation exist. It will also clarify whether they bear any common structural similarities, answering questions on species differences, different proteins exerting the same in-vitro activity or different purification procedures. From a physiological point of view the isolation of GnSAF from FF would be of significance in the field of human fertility.
In conclusion, the present paper describes a purification procedure leading to electrophoretic homogeneity for the GnSAF factor from human follicular fluid and will enable further studies of the structure and physiology of this substance.
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Acknowledgments |
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Notes |
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6 To whom correspondence should be addressed
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References |
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Submitted on October 29, 1998; accepted on March 2, 1999.