Gonadotropin Surge-Attenuating Factor Bioactivity in Serum from Superovulated Women Is Not Blocked by Inhibin Antibody'

Transcription

1 BIOLOGY OF REPRODUCTION 52, (1995) Gonadotropin Surge-Attenuating Factor Bioactivity in Serum from Superovulated Women Is Not Blocked by Inhibin Antibody' B. BYRNE, 2 ' 3 P.A. FOWLER, 3 M. FRASER, 3 M.D. CULLER, 4 and A. TEMPLETON 3 Department of Obstetrics and Gynaecology, 3 University of Aberdeen, Foresterhill Aberdeen AB9 2ZD, United Kingdom Immunobiology Research Institute, 4 Annandale, New Jersey ABSTRACT Primary pituitary cultures from adult female rats were used to investigate gonadotropin surge attenuating factor (GnSAF) bioactivity. Serum from superovulated women was charcoal-treated and incubated with either an inhibin antibody (MC4), normal sheep serum (NSS), or serum-free defined medium (SFDM) before addition to cell culture. The reduction in GnRH-induced LH secretion (GnSAF bioactivity) produced by the serum ( %, p <.1, of control) was not altered by prior incubation with either MC4 or NSS ( and %, p <.1, of control, respectively). This indicates that GnSAF bioactivity present in serum from superovulated women is not entirely attributable to inhibin. Recombinant human inhibin reduced basal FSH secretion to 24.6 ± 4.7% (p <.1) of the control value. However, this was totally abolished by prior incubation with MC4, but not NSS. We have also shown that ovarian steroids are not responsible for GnSAF bioactivity in vitro. In conclusion, in contrast to findings in superovulated rats, inhibin antibody did not block GnSAF bioactivity in serum from superovulated women. INTRODUCTION Over recent years, new information has come to light regarding the control of gonadotropin secretion. Schenken and Hodgen [1] showed that intact, but not ovariectomized, monkeys injected with purified FSH had reduced GnRHinduced LH and FSH secretion, resulting in inhibition of the LH surge. It was therefore suggested that the ovaries produced a factor, gonadotropin surge inhibiting factor (GnSIF), that caused a reduction in gonadotropin secretion. A similar effect was seen in women superovulated for IVF [2], and Messinis and Templeton [3] gave this factor the name gonadotropin surge attenuating factor (GnSAF). It is probable that GnSAF and GnSIF are the same factor [4, 5]. Pituitary responsiveness to GnRH is reduced by GnSAF [6], the release of which is stimulated by FSH in both women [7] and rats [8]. Both serum and follicular fluid (hff) from superovulated women have been shown by means of an in vitro ovine pituitary bioassay to contain GnSAF bioactivity [9, 1]. The presence of GnSAF bioactivity has also been demonstrated in serum from spontaneously cycling women by the suppression of GnRH-induced LH secretion from rat pituitary monolayers [11]. Both Danforth et al. [12] and Fowler et al. [9] removed inhibin by heparin chromatography of porcine follicular fluid (pff) or hff and found that the unbound fractions still contained GnSAF bioactivity. Fowler et al. [13] have also shown, using ultrafiltration, that GnSAF bioactivity is distinct from inhibin. Accepted August 24, Received June 15, 'B. Byrne is supported by a University of Aberdeen Medical Endowments PhD studentship. PA Fowler is supported by Scottish Hospital Endowments Research Trust (SHERT) grant Correspondence. FAX: Recently, however, Culler [14] demonstrated that immunization against inhibin reversed the FSH-induced increase in GnSAF bioactivity in cycling rats. Culler [14] used an anti-human inhibin a-subunit serum (MC4) generated in ewes. This, injected into superovulated rats, abolished the suppression of GnRH-induced LH and FSH secretion observed in superovulated rats injected with a normal sheep serum (NSS). From this study Culler concluded that under some circumstances inhibin was responsible for GnSAF bioactivity in rats or that it might be involved in a multifactor gonadotropin surge-suppressing cascade. Therefore, the aim of the present study was to investigate the effects of the inhibin antibody that Culler used [14] on GnSAF bioactivity in serum and hff from superovulated women and also to assess the biopotency of the inhibin antibody in vitro via a rat pituitary bioassay based on the method reported by Danforth et al. [12]. In this paper we present data showing that in vitro, the inhibin antibody has no effect on GnSAF bioactivity expressed in terms of GnRH-induced LH secretion from rat pituitary monolayer cultures. MATERIALS AND METHODS Preparation of Rat Anterior Pituitary Cell Monolayers Adult female Sprague-Dawley rats were maintained under a constant 12L:12D, 22 C environment with ad libitum access to food and water. For each cell culture 15 rats, selected at random during the estrous cycle, were killed by stunning and cervical dislocation. Dispersion and culture of the pituitary cells were carried out through use of the protocol described by Fowler et al. [15]. on 18 December

2 INHIBIN ANTIBODY DOES NOT BLOCK GnSAF BIOACTIVITY 89 Briefly, the pituitaries were removed from 15 rats and transferred to Dulbecco's PBS (DPBS) containing Ca 2+ and Mg 2+, 5% (v:v) penstrep, and 5 p1 gentamicin/l (Sigma Chemical Co,, Poole, Dorset, UK). All further procedures were carried out under sterile conditions in a flow cabinet. After removal of the neurointermediate lobes, posterior pituitary, and vascular tissue, the pituitaries were quartered and stirred in 13 ml of DPBS containing 1% BSA, 2% glucose, and 24 mg of Sigma T-8253 trypsin (Sigma Chemical Co.) for 3 min in a 5% CO 2 incubator at 37 C. The pituitary fragments were then washed in serum-free defined medium (SFDM;.1% BSA fraction V, 1 jig transferrin/ml,.5% insulin, 5% penstrep, and 5 1 gentamicin/l added to stock DME/F-12 [Sigma Chemical Co.]); they were then transferred to a sterile container and mechanically dispersed with the aid of siliconized pasteur pipettes. The cell suspension was spun at 4 x g for 8 min at 22 C and then resuspended in SFDM. After the cell suspension had been spun and resuspended a second time, viability was assessed through use of trypan blue exclusion. Preparations of < 7% viability were discarded. Primary pituitary cell cultures were at 1 viable cells/ 5 LAI culture medium per well in 24-well tissue culture plates. The cells were cultured with SFDM under sterile conditions for 48 h at 37 C in a water-saturated atmosphere of 5% CO2:95% air. Medium and nonattached cells were removed by aspiration and discarded. All experiments were then carried out on quadruplicate wells as follows: 45. l 1 of fresh SFDM was added together with the treatments, which were made up to 5 Rl with SFDM. All the culture plates contained at least four control wells receiving SFDM only. After 48-h incubation with the test substances, the medium was collected and stored at -2 C (basal secretion). The cells were then treated with.1 Rpmol GnRH/L (Fertagyl; Intervet UK Ltd., Cambridge, UK). After a 4-h incubation the medium was collected and stored at -2 C (GnRH-induced secretion). Treatment of Serum and hff Serum and hff were obtained from normally cycling women who had been superovulated for IVF in a protocol approved by the Joint Ethical Committee of Aberdeen. The women were superovulated in a short protocol comprising 7-1 days of norethindrone followed by Buserelin (Hoescht UK Ltd., Hainslow, Middlesex, UK) and a decreasing daily dose of human post-menopausal gonadotropin (Pergonal; Serono Laboratories Ltd., Welwyn Garden City, UK) totaling 25-4 amps per woman per cycle. The serum was collected h before recovery of oocytes; at the time of recovery the hff was aspirated. Serum and hff were each pooled from 32 women. The mean number of follicles per woman was , and mean follicle diameter was mm. The serum and hff were subjected to two-fold charcoal treatment to remove steroids as described by Fowler et al. [9]. At a dose of 5,l/well the steroid content of the hff was reduced from 15 to.95 nmol/l estradiol and from 2,7 to.174 nmol/l progesterone. Similarly, the steroid content of the serum was reduced from.76 to.2 nmol/l estradiol and from.38 to.15 nmol/l progesterone. This process also reduces dimeric inhibin immunoreactivity by 3. ± 5.5% [15]. Generation of the Inhibin Antibody Ovine anti-human inhibin a-subunit serum MC4 was generated and characterized as described by Culler and Negro-Vilar [16]. Briefly, an immunogen made of a conjugate of the synthetic 1-32 amino acid sequence of the oa-chain of human inhibin (INI 32 ; Peninsula Labs., Belmont, CA) and human serum albumin (hsa; Sigma Chemical Co., St. Louis, MO) was emulsified with Freund's complete adjuvant (Sigma) for the primary inoculation, which was injected into mixed Dorset-Hampshire ewes. Secondary injections consisted of the conjugate emulsified with Freund's incomplete adjuvant; these were given 1 days after the primary inoculation and at monthly intervals thereafter. The ewes were bled via the jugular to give the MC4, which contained 84.9 mg protein/mi. The NSS was obtained from nonimmune sheep, sterilized, and then made up in PBS (81. mg protein/ml) to a protein concentration similar to that of the MC4. Recombinant Human Inhibin Recombinant human inhibin was generously provided by Dr. DM. de Kretser and Biotech Australia Pty. Limited (Roseville, Australia). On receipt, 1-ql aliquots of 5 ng/ml stock solution in sterile distilled water were stored at -4 C. This material was diluted in of SFDM immediately before dilution to doses of.25,.25,.25, and 2.5 ng/well in 5-pl aliquots added to cell culture. Experiment 1: GnSAF Bioactivity in Serum and hfffrom Superovulated Women The serum and hff were added to two separate cell cultures at doses between.5 and 5 pj in quadruplicate wells. Experiment 2: Effects of Recombinant Human Inhibin, MC4, and NSS on Gonadotropin Secretion Recombinant human inhibin (doses between.25 and 2.5 ng/well) and MC4 and NSS (doses between.5 and 5 li/well, 4 ng-4 mg protein/well) were added to two separate cell cultures in quadruplicate wells. Experiment 3: Effect of Inhibin Antibody on GnSAF Bioactivity in Serum and hff from Superovulated Women and Response to Recombinant Human Inhibin The inhibin antibody, MC4, and NSS were diluted 1:1 in the serum-free defined medium. A dose (5 R1l) of superovulated serum that was maximally effective (in terms of reducing GnRH-induced LH secretion) or a submaximal on 18 December 217

3 . 9 BYRNE ET AL. t a 14 ' 12 ' 1 Mla 8 r. o 6 - a) # Experiment 5: Effects of Steroid Hormones Water-soluble progesterone and estradiol (Sigma Chemical Co.) were added to quadruplicate wells to two separate rat pituitary cell cultures at doses between.1 and 1 nmol/l. Estradiol and progesterone were also added to pituitary monolayers in combined doses (.1-1 nmol/ L) of each steroid in a two-way titration. o 2 ce r. e n V I4A h 1U/ I I I I I gl superovulated serum/well I I I.5.5 xil hff/well 5 5 ED 5 = 1.8 Il/well ED 5 =.57 l/well ED 5 = 26. I.l/well FIG. 1. Dose-dependent effects of (a) serum and b) follicular fluid, from superovulated women, on GnRH-induced LH (closed circles) and basal FSH (open circles) secretion from rat pituitary monolayers. Combined data from two experiments are plotted; n = 2 x 4. Significance values are compared with control gonadotropin secretion from the same culture dishes (AN- OVA): = p <.5, = p <.1, = p <.1. dose (5 Rl) of hff from superovulated women and recombinant inhibin (2.5 ng/well) were incubated with 1:1 dilutions of MC4 or NSS, or with medium alone, at a ratio of 1:1 (v:v) for 2 min at room temperature before addition to cell culture. Different doses of hff and serum were used to equalize the concentration of inhibin present (hff = 74.8 ng/ml inhibin and superovulated serum = 7.5 ng/ml inhibin). For controls, MC4 and NSS were added alone to culture. A 5-1l combined dose of each of the test substances was added to two separate cell cultures in quadruplicate wells. The final quantities added to the cell culture were.2 mg protein/well of MC4 and NSS (= 2.5 RIl/well), 1.25 ng/well of inhibin, and 25 Rl/well of hff or serum. Experiment 4: Quantification of Inhibin Antibody Biopotency Using Recombinant Human Inhibin Doses of both MC4 and NSS between.25 and 25 pxl/ well were added to two separate cell cultures after prior incubation with the same dose of 1.25 ng recombinant inhibin/well used in experiment 3. RAs Concentrations of LH in GnRH-treated, and FSH in basal, cell-conditioned media were determined through the use of homologous rat RIAs. Sensitivities and intraassay and interassay coefficients of variation for these RIAs were.6 ng/ ml FSH (NIDDK-rFSH-RP-3) using NIDDK-anti-rFSH-S11 and 6.5% and 14.6%, respectively, and.3 ng/ml LH (NIDDKrLH-RP-2) using NIDDK-anti-rLH-S11 and 7.1% and 16.5%, respectively. Commercial kits were used to determine estradiol and progesterone concentrations in hff and serum as previously described [9]. Dimeric inhibin immunoreactivities were determined by immunoradiometric assay [17]; the coefficients of variation were 5.% and 7.%, respectively. Statistical Analysis Pituitary responses to the test substances are expressed as percentages of the relevant control gonadotropin concentrations secreted from wells on the same culture plates. This process permitted data from duplicate cultures to be combined. The differences within whole experiments and any dose-dependent effects were assessed by one-way AN- OVA following log transformation to normalize variances. Specific differences between controls and test substances were assessed by Dunnett's multiple comparison. Dose-response curves were also compared by calculating the ED 5 s from the combined duplicate data. The ED 5 s were determined by establishing 5% of the maximal effect (greatest suppression seen during the cell cultures composing this study) of the dose-response curves to be compared and interpolating from log-linear regression equations for each dose-response curve. Values are presented as means - SEM. RESULTS Experiment 1: GnSAF and Inhibin Bioactivity in Serum and hff from Superovulated Women Both serum (Fig. la) and hff (Fig. lb) from superovulated women contained GnSAF bioactivity that reduced GnRH-induced LH secretion in a dose-dependent manner, to 26.5 ± 7.% (p <.1) and 55.9 ± 5.2% (p <.1) of control levels, respectively, at the 5 Rl/well dose. The serum ED 5 for reduction in GnRH-induced LH secretion was much lower (> 1-fold difference) than that of the hff. The hff reduced basal FSH secretion in a dose-dependent manner ( % p <.1), but became ineffective on 18 December 217

4 INHIBIN ANTIBODY DOES NOT BLOCK GnSAF BIOACTIITY 91 at the 5 ll/well dose; however, the serum stimulated basal FSH after suppression at the.5 Rl/well dose. Experiment 2: Effects of Recombinant Inhibin, MC4, and NSS on Gonadotropin Secretion Recombinant human inhibin reduced both GnRH-induced LH secretion and basal FSH secretion in a dose-dependent manner to % (p <.5) and % (p <.1) of control values, respectively (Fig. 2a). The maximally effective dose was 2.5 ng/ml inhibin. In contrast, NSS and MC4 added alone to cell culture caused no suppression of GnRH-induced LH secretion, although the MC4 caused significant stimulation of basal FSH secretion to % (p <.1) of control values at 5 ll/well (Fig. 2c). Experiment 3: Effect of Inhibin Antibody on GnSAF Bioactivity in Serum from Superovulated Women and Recombinant Human Inhibin When NSS or MC4 was added to the cell culture in combination with the serum from superovulated women, there was attenuation of GnRH-induced LH secretion to % (p <.1) and % (p <.1) of control values, respectively (Fig. 3a). This was not significantly different from the attenuation caused by the serum alone. None of the treatments used had any significant suppressive effect on basal FSH secretion (Fig. 3b). A submaximal dose (5 Il/ well) of the hff from superovulated women reduced GnRHinduced LH secretion to % (p <.1) of control values (results not shown). Prior incubation of the hff with the 1:1 dilution of MC4 had no effect on this suppression; in this case, GnRH-induced LH secretion was reduced to % (p <.1) of the control level. However, while basal FSH secretion was reduced to 55.6 ± 5.4% (p <.1) of the control value by the hff alone, prior incubation of the hff with MC4 significantly reduced inhibin bioactivity (basal FSH secretion reduced to % of control, p <.1). When the recombinant human inhibin and MC4 were incubated together before being added to cell culture, there was complete abolition of the suppressive effects of inhibin on basal FSH secretion relative to that for the control (Fig. 3d). However, when the 1.25 ng/well dose of recombinant human inhibin was incubated with NSS, the suppression of basal FSH by the recombinant inhibin to % (p <.5) of the control value was maintained (Fig. 3d). None of the treatments shown in Figure 3c had statistically significant effects on GnRH-induced LH secretion. However, the 2% suppression of GnRH-induced LH secretion resulting from the addition of recombinant human inhibin was totally removed by MC4 but not NSS. Experiment 4: Quantification of Inhibin Antibody Biopotency It should be noted that in this experiment, control was taken to be the equivalent dose of NSS or MC4 to com- r. Ib Im IOU I 1 I 8 To 6 1Q! '. 4.c 12 8 C 6 8, 4 a) o o Co o I I - -v L 'An 14C ne 1, "^ 16(U b) g 14 ~V C c., 16t r- I- l- I- l- l- ng inhibin/well I I I I I , gi NSS/well I I I I gi MC4/well ED 5 =>25.ng/well ED 5 =.7ng/well FIG. 2. Dose-dependent effects of (a) recombinant human inhibin, (b) NSS, and (c) the inhibin antibody MC4 on GnRH-induced LH (closed circles) and basal FSH (open circles) secretion. Combined data from two experiments are plotted; n = 2 x 4. Significance values are compared with control gonadotropin secretion from the same culture dishes (ANOVA): = p <.1, = p <.1. pensate for nonspecific effects of MC4 in particular on basal FSH secretion. When the MC4 was incubated with the recombinant inhibin, the.25 pl/well dose significantly altered, and the l/well doses totally abolished, the on 18 December 217

5 92 BYRNE ET AL. Experiment 5: Lack of Effect of Steroids As can be seen in Figure 5, addition of estradiol and progesterone singly produced no significant change in gonadotropin secretion. In combination they produced slight, but significant, stimulation (p <.5) of both GnRH-induced LH secretion (to % of control values at a dose of 1 nmol/l) and basal FSH secretion (to % of control values at a dose of 1 nmol/l). FIG. 3. The effects of.2 mg/well protein (2.5 il/well) of the inhibin antibody (MC4) and NSS on GnSAF and inhibin bioactivities. Shown are the effects of a final dose of 25 l/well of serum from superovulated women (SS) on (a) GnRH-induced LH and (b) basal FSH secretion and a final dose of 1.25 ng/well of recombinant inhibin on (c) GnRH-induced LH secretion and (d) basal FSH secretion in the presence or absence of MC4 and NSS. The results are expressed relative to MC4 + SFDM as 1%. Combined data from two experiments are plotted; n = 2 x 4. Italicized letters denote significance values (p <.5) compared with values for either (a) MC4 + SFDM, (b) serum from superovulated women, or (b) recombinant inhibin + SFDM on gonadotropin secretion from the same culture dishes (ANOVA). suppressive effect of the recombinant inhibin on basal FSH secretion (Fig. 4a). The lowest dose (from Fig. 4a) to totally abolish the effects of the inhibin on basal FSH secretion was.25 p.l/well (9 ng protein/well) per 1.25 ng recombinant inhibin/well. The MC4 also totally abolished the suppressive effects of this dose of recombinant inhibin on GnRH-induced LH secretion. Since serum from superovulated women contained.19 ng/well (7.5 ng inhibin/ml) at a dose of 25 p.l/well, the inhibin bioactivity would have been totally removed by incubation with the 2.5 p.l/well dose of MC4 used in experiment 3. When l amounts of NSS (Fig. 4b) were incubated with a dose of 1.25 ng recombinant inhibin/well, the resulting doses of pl/well slightly potentiated the suppressive effects of the inhibin on basal FSH and GnRH-induced LH, by between 21 and 27% of secretion in the presence of an equivalent dose of NSS alone. DISCUSSION In the present investigation we have demonstrated, in agreement with other studies [9,1,18], the presence of GnSAF bioactivity in both serum and hff from superovulated women. As reflected by the ED 5 s, the GnSAF bioactivity is markedly different between the serum and the hff. This may be attributable to the hff's being aspirated during oocyte recovery, 36 h after hcg injection according to the 1VF protocol. Fowler et al. [15] have shown that the maximum amount of GnSAF is produced by small follicles. Since the hcg injection induces final maturation of these follicles it may possibly reduce the amount of GnSAF being secreted into the follicular fluid. Interestingly the serum from superovulated women had stimulatory effects on basal FSH secretion. The reason for this is unclear, although it may be that this particular batch of serum has high concentrations of activin. The ED 5 for activin-stimulated FSH secretion from rat pituitary cultures is comparable to that for inhibin [19]. Although inhibin masks the effects of activin [2] and follistatin binds activin [21], it is possible that rat pituitary cells are highly sensitive to activin in serum and hff from superovulated women. A final possibility is simply that of nonspecific protein-loading effects. Using our ovine pituitary models [22], we have shown that the same batch of superovulated serum as that used in the present study markedly suppresses ovine pituitary responsiveness to GnRH. This was seen both in the pituitary monolayer system, which includes 12.5% of sera in the buffer, and in the pituitary perifusion model, where heat-inactivated plus charcoaltreated serum from non-superovulated humans was used to control for nonspecific effects. We have clearly shown that, contrary to Culler's findings for rats in vivo, in vitro GnSAF bioactivity is not blocked by the inhibin antibody MC4. The suppression of GnRH-induced LH secretion (by more than 7% in relation to the control) caused by the serum in vitro was not abolished by the inhibin antibody. This leads us to conclude that the GnSAF bioactivity present in serum from superovulated women and detected by this in vitro bioassay is not attributable to inhibin. We have shown that MC4 and NSS do not cause any suppression of either basal FSH or GnRH-induced LH secretion, although there is a significant stimulation of gonadotropin secretion caused by the MC4. This may be due to endogenous activin activity present in the MC4 that is free to act because of the presence of antibodies in the serum that bind endogenous inhibin. Furthermore, recombinant human inhibin is capable of suppressing both basal on 18 December 217

6 INHIBIN ANTIBODY DOES NOT BLOCK GnSAF BIOACTMTY 93. go a 4 c = saz 4-1 An I IL'L I A/ 14U ! I"h - U) inhibin I I I I I I I I C-' {.{1 JL'L/WCII ~~~ ~.. AfffA.-only I I I I p1 NSS/well P 25 inhibin only FIG. 4. Dose-dependent effects of (a) NSS and (b) MC4 on the actions of 1.25 ng recombinant inhibin/well on GnRH-induced LH (closed symbols) and basal FSH (open symbols) secretion. In this figure the gonadotropin secretion is shown relative to the effects of equivalent doses of either MC4 or NSS at 1% as appropriate. GnRH-induced LH and basal FSH secretion in the presence of 1.25 ng recombinant inhibin/well alone (without either MC4 or NSS) are shown by bars labeled "inhibin only" while GnRH-induced LH and basal FSH secretion in the absence of recombinant inhibin are shown by bars reaching 1% on the left of the abscissas. Combined data from two experiments are plotted; n = 2 x 4. Significance values are compared with gonadotropin secretion in the presence of the equivalent dose of MC4 or NSS alone from the same culture dishes (ANOVA): = p <.5, = p < : U 4-4- cu IOU, / r IOU An V i -I IOU It A I U I1 M u! - I I I I I I I I I I c) I L I I I I FSH and GnRH-induced LH secretion in our hands; this agrees with findings from other studies [23]. In order to check the effectiveness of the inhibin antibody we incubated it with recombinant human inhibin. Not surprisingly, dose-dependent and marked suppression of basal FSH secretion caused by recombinant human inhibin was blocked by prior incubation with MC4. This effect of MC4 is specific, since NSS did not reduce recombinant human inhibin 6 I I I I I I I I I Steroids (nmol/well) 1 FIG. 5. The effects of (a) estradiol, (b) progesterone, and (c) estradiol and progesterone combined on both GnRH-induced LH (closed circles) and basal FSH (open circles) secretion. Combined data from two experiments are plotted; n = 2 x 4. Significance values are compared with control gonadotropin secretion from the same culture dishes (ANOVA): = p <.5. on 18 December 217

7 94 BYRNE ET AL. bioactivity. The results for GnRH-induced LH secretion followed a similar pattern, demonstrating that any suppressive effect caused by the recombinant inhibin was overcome by the inhibin antibody. These results show that the inhibin antibody, MC4, is capable of blocking inhibin bioactivity under the conditions used in the present study. This supports the observations reported here and those from earlier studies demonstrating that inhibin does not account for all GnSAF bioactivity. The biopotency for the inhibin antibody was determined by incubating a range of doses of both MC4 and NSS with a maximally effective dose of recombinant inhibin. From this experiment we were able to validate the effects of 2.5 Il/well of MC4 on inhibin bioactivity as examined in experiment 3. While MC4 stimulated basal FSH, the stimulation was by < 2% at the 2.5 l/well dose used in experiment 3, in which the effects of superovulated serum, hff, and recombinant human inhibin on basal FSH and GnRH-induced LH secretion were totally abolished. We have also demonstrated in the current study that steroids present in either the test substances, NSS, or MC4 are not responsible for the GnSAF bioactivity measured through use of rat pituitary monolayers. Although there was stimulation of both basal FSH and GnRH-induced LH secretion by the addition of combined doses of estradiol and progesterone to cell culture, this occurred at a concentration of between 1 and 1 nmol/l. After charcoal treatment, steroid concentrations in the serum from superovulated women were less than.1 nmol/well. Similarly, the steroids in the MC4 and NSS were <.2 nmol/l estradiol and < 1 nmol/l progesterone, and even the maximum dose of 5 p.l/well entailed further dilution of 1:1 or more. The present study demonstrates that GnSAF bioactivity in serum and hff from superovulated women is not attributable to inhibin. However, we have not shown that inhibin is not responsible for GnSAF bioactivity in rats. It may be that in rats inhibin and GnSAF are the same, although the fact that rat pituitary cells are very sensitive to GnSAF bioactivity suggests that this is not the case. Therefore, the question this study raises is why the inhibin antibody, MC4, effectively blocks GnSAF bioactivity in rats in vivo but not GnSAF effects on rat pituitary cells in vitro. While it may be that the steroid milieu of the pituitary cells and the stage of the estrous cycle have important influences on the responsiveness of these cells to GnSAF bioactivity in vivo, answering this question will necessitate further carefully designed studies. It is also important to note that the results presented here reveal GnSAF bioactivity only as defined by the in vitro bioassay. It is not yet clear how these results relate to suppression of the preovulatory LH surge in vivo by GnSAF. In conclusion, although the inhibin antibody MC4 is capable of blocking GnSAF bioactivity in superovulated rats in vivo, it is not capable of blocking the GnSAF bioactivity present in serum from superovulated women. Therefore the GnSAF bioactivity in serum from superovulated women is due to neither the ovarian steroids nor inhibin. ACKNOWLEDGMENTS The recombinant inhibin was generously provided by Biotech Australia Pry Ltd., 28 Barco Rd., East Roseville, NSW, 269 Australia. We thank the NLkADDK (Baltimore) and SAPU (Carluke Hospital Lawarkshire, Scotland, ML8 5ES) for the RIA material. We are grateful to the staff of the Medical School Animal House (University of Aberdeen, Scotland, AB9 2ZD) for maintaining the rats used in this study. We also thank Dr. P. Knight, Department of Biochemistry and Physiology, University of Reading, Whiteknights, Reading, RG6 2AJ, UK, for dimeric inhibin immunoreactivity measurement. REFERENCES 1. Schenken RS, Hodgen GD. Follicle-stimulating hormone induced ovarian hyperstimulation in monkeys: blockade of the luteinising hormone surge. J Clin Endocrinol & Metab 1983; 57: Messinis IE, Templeton A, Baird DT. Relationships between characteristics of endogenous luteinising hormone surge and the degree of ovarian hyperstimulation during superovulation induction in women. Clin Endocrinol 1986; 25: Messinis IE, Templeton A. In vivo bioactivity of gonadotrophin surge attenuating factor (GnSAF). Clin Endocrinol 199; 33: Messinis IE, Templeton AA. Evidence that gonadotrophin surge-attenuating factor exists in man. J Reprod Fertil 1991; 92: Balen AH, Jacobs HS. Gonadotrophin surge attenuating factor: a missing link in the control of LH secretion. Clin Endocrinol 1991; 35: Messinis IE, Templeton A. Superovulation induction in women suppresses luteinising hormone secretion at the pituitary level. Clin Endocrinol 199; 32: Messinis IE, Lolis D, Papadopoulos L, Tshalina Th, Papanikolaon N, Seferiadis K, Templeton Ak Effect of varying concentrations of follicle stimulating hormone on the production of gonadotrophin surge attenuating factor (GnSAF) in women. Clin Endocrinol 1993; 39: Koppenaal DW, Tijssen AMI, van Dieten JAMJ, de Koning J. The self-priming action of LHRH is under negative FSH control through a factor released by the ovary: observations in female rats in vivo. J Endocrinol 1991; 129: Fowler PA, Messinis IE, Templeton AA. Inhibition of LHRH-induced LH and FSH release by gonadotrophin surge-attenuating factor (GnSAF) from human follicular fluid. J Reprod Fertil 199; 9: Fowler PA, Templeton A, Messinis IE. The ovarian modulation of GnRH-induced LH secretion in women. Hum Reprod 1993; 8(suppl 2): Byrne B, Fowler PA, Messinis IE, Templeton A. 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