Blockade of the estrogen-induced luteinizing hormone surge in monkeys: a nonsteroidal, antigenic factor in porcine follicular fluid *

Transcription

1 FERTILITY AND STERILITY Copyright 1984 The American Fertility Society Vol. 41, No.1, January 1984 Printed in U.SA. Blockade of the estrogen-induced luteinizing hormone surge in monkeys: a nonsteroidal, antigenic factor in porcine follicular fluid * Victoria M. Sopelak, Ph.D. Gary D. Hodgen, Ph.D.t Pregnancy Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland That both follicular fluid extracts and ovarian hyperstimulation block estrogeninduced gonadotropin surges in primates has been established. In the present study, the differential actions of gonadotropin-releasing hormone (GnRH) and estradiol benzoate (E2B) on bioassayable LH (BIO-LH) secretion in the presence of a porcine follicular fluid (pff) extract were assessed. Further, the inherent immunogenicity of the follicular fluid factor(s) that block estrogen-induced surges was examined. In castrated monkeys, administration of Florisillcharcoal-extracted pff did not affect BIO-LH secretion induced by exogenous GnRH, although the pff extract abolished E2B-induced BIO-LH surges. In monkeys given pff chronically (for 6 to 8 months), immune responses capable of neutralizing the inhibitory actions of pff on the E2B-induced BIO-LH surges developed. In these pff-immunized monkeys, the BIO-LH surge induced by an E2B challenge was restored despite concurrent pff injections. Fertil Steril41 :108, 1984 Clinical therapies utilizing human menopausal gonadotropins (hmgs) for stimulation of the natural ovarian cycle have shown that irrespective of the duration of treatment, the dose ofhmg given, or concentrations of estrogen attained during these cycles, spontaneous luteinizing hormone (LH) surges seldom occur. 1 Similarly, monkeys stimulated with "pure" follicle-stimulating hormone (FSH) or hmg demonstrate a blockade of estrogen-positive feedback for the LH surge in the presence of hyperstimulated ovaries. 2-4 We have suggested that some factor(s) may be secreted by Received April 19, 1983; revised and accepted August 11, *Supported in part by Ford Foundation grant treprint requests: Dr. Gary D. Hodgen, Pregnancy Research Branch, Building 18, Room 101, NICHD/NIH, Bethesda, Maryland exogenously stimulated ovaries, which can block the estrogen-induced LH surge. Porcine follicular fluid (pff) may contain constituents similar to those produced by hyperstimulated ovaries, in that pff also blocks estradiolinduced gonadotropin surges. Specifically, when charcoal-extracted pff is administered to intact monkeys just prior to the expected spontaneous midcycle LH/FSH surges, ovulation is delayed or abolished. 5 When given during the early follicular phase, pff extract blocks the LH/FSH surges induced by estradiol benzoate (E 2B) in intact monkeys.6 Collectively, this evidence indicates that a nonsteroidal ovarian factor(s), whether imposed by injection of follicular fluid or gonadotropin-induced ovarian hyperstimulation, can modulate gonadotropin secretion. We conducted the following studies to assess how pff may affect both gonadotropin-releasing 108 Sopelak and Hodgen Gonadotropin surge inhibiting factor Fertility and Sterility

2 - ~~ --~-~---~-~ ~----' "" hormone (GnRH) and E2B-induced bioassayable LH (BIO-LH) secretion. Using castrated female monkeys, we determined (1) whether a crude pff extract desensitized the pituitary to acute GnRHenhanced BIO-LH secretion; (2) whether the enhancement ofbio-lh secretion effected by GnRH or E2B-positive feedback could be differentially affected by pff; and (3) whether pff factors influencing LH secretion may be inherently immunogenic. Castrated monkeys were utilized in order to study the effects of pff without the potential confounding effects of ovarian feedback. MATERIALS AND METHODS STUDY I-pFF EFFECTS ON GnRH AND E.B RESPONSES Long-term ovariectomized female rhesus monkeys (n = 8) were chronically cannulated via the femoral vein and placed in a vest and mobile tether apparatus. 7 On day 1, four of these monkeys (without prior pff exposure) were injected subcutaneously at 8:00 A.M. with 10 ml of Florisillcharcoal-extracted pff (Supelco, Inc., Bellefonte, PA), then 5 ml daily at 8:00 A.M. for 9 days. The combined Florisillcharcoal extraction was employed to obtain a pff-product having very low steroid levels (estradiol, 19 pg/ml; progesterone, 0.2 ng/ml; testosterone, 20 ng/di). Control monkeys were similarly treated with saline. On day 3, both control and pff-treated monkeys were serially injected intravenously with 50 jl.g of GnRH at noon, 2:00, 4:00, and 6:00 P.M.; plasma was collected at 0, 15, 30, 60, and 90 minutes following each GnRH injection. At noon on day 6, all monkeys were injected with 50 jl.g/kg E2B; plasma was collected at 2-hour intervals over the ensuing 96 hours from these unanesthetized monkeys. STUDY 2---IMMUNOGENICITY OF pff Long-term ovariectomized female rhesus monkeys were assigned to three groups: (1) control, saline-treated monkeys (n = 4); (2) pff -treated monkeys (n = 3) that had never before received pff; and (3) pff-immunized monkeys (n = 4). Antigenic responses were developed in the castrated monkeys (group 3) by injecting them with 10 ml of sterile filtered pff extract subcutaneously once monthly for 6 to 8 months. Four weeks after the last pff injection, these immunized monkeys (group 3) and castrated monkeys lacking prior pff exposure (nonimmunized; group 2) were injected subcutaneously at 8:00 A.M. (day 1) with 10 ml of Florisillcharcoal-extracted pff, then 5 ml daily at 8:00 A.M. for 5 days. Control animals (group 1) received saline. At noon on day 3, monkeys in all three groups received 50 jl.g/kg E2B; serum was collected by venipuncture under ketamine (Parke-Davis, Morris Plains, NJ) anesthesia at 6-hour intervals for 96 hours. Ouchterlony diffusion plates were run to test the immunoreactivity of serum from the four pff-immunized castrated monkeys (group 3) versus pff and pig serum. Serum from two castrated monkeys immunized to pig serum as well as saline-treated castrated monkeys were similarly tested on Ouchterlony gels. Plasma or serum samples from both experiments were stored frozen until measurement of E2 and in vitro BIO-LH using testosterone production by dispersed mouse testis as the end point, as previously described. 8 -u Data were compared using a one-way analysis of variance to determine whether responses to GnRH or E2B were different between saline-treated control monkeys and pff-treated monkeys, with and without immunization. RESULTS STUDY I-pFF EFFECTS ON GnRH AND E.B RESPONSES In the saline-treated control castrated monkeys, each injection of GnRH was followed by a significant (P < 0.05) elevation ofbio-lh (fastigium, 42.2 ± 4.4 jl.g/ml; mean ± standard error of the mean [SEM]) above the baseline on day 3 (13.2 ± 5.7 jl.g/ml); obvious nadirs were observed 2 hours after each GnRH injection (32.2 ± 8.1 jl.g/ml) (Fig. 1), although circulating LH levels remained well above the initial baseline. Indeed, all BIO-LH responses to serial GnRH injection were comparable (P > 0.05). Similarly, pfftreated nonimmunized castrated monkeys demonstrated significant (P < 0.05) elevations ofbio LH in blood (fastigium, 48.4 ± 2.0 jl.g/ml, and baseline, 21.5 ± 3.6 jl.g/ml, respectively). Again, nadirs were apparent 2 hours after each GnRH injection (35.5 ± 3.8 jl.g/mi) (Fig. 2). Compared with saline-treated castrated monkeys, the GnRH-induced gonadotropin responses of the pff -treated nonimmunized castrated monkeys were not different (P > 0.05). Baseline values of BIO-LH on day 1, prior to any treatment, were Vol. 41, No.1, January 1984 Sopelak and Hodgen Gonadotropin surge inhibiting factor 109

3 Onj" "T" "T" GT" : 622 H! Saline Treated :~!li\ 20: I :! 10 : i :! I i :! ~ 3 6 iii "T" T or T, \ 618 H 100 'l Sal;.. T 9D i : 80 : t! 70! I \ Iii : il \iri\f\ I, ~ i l6 V~,. I I!! 30! : '" G"fH : 624 H! Satine Treated NOON 2PM 4PM &PM IPM NOON 2PM 4PM IPM 8PM Figure 1 Composite patterns of BIO-LH in plasma of control castrated monkeys receiving 50 ij.g GnRH at noon, 2:00, 4:00, and 6:00 P.M. on day 3. Broken lines represent estimates of values above the detection limits of the assay. similar in both groups (saline-treated, 36.2 ± 6.2 fj.g/ml; pff -treated, 44.8 ± 8.4 fj.g/ml). Irrespective of the GnRH treatment on day 3, the saline-treated castrated monkeys responded to the E2B injection on day 6 with a typical biphasic response ofbio-lh secretion (Fig. 3): BIO LH levels decreased wi thin 12 hours following the E2B challenge from 14.7 ± 4.7 fj.g/ml to 1.8 ± 0.2 fj.g/ml). These nadirs were followed by a fastigium (36.6 ± 11.1 fj.g/ml), which occured 53 ± 6 hours after the E2B injection. In contrast, pff -treated nonimmunized castrated monkeys did not manifest BIO-LH surges (Fig. 3) in response to an equivalent E2B challenge. Although these E2B treatments caused a decrease in the basal secretion of gonadotropins within 12 hours to nadirs similar (P > 0.05) to those found in saline-treated control monkeys, these lower LH concentrations persisted throughout the remainder of the study. Thus, pff-treated monkeys responded to the GnRH like saline control monkeys but were unresponsive to the positive feedback effects of estrogen on pituitary LH release. STUDY 2-IMMUNOGENICITY OF pff Since our pff extract had not affected the BIO LH response to GnRH, we assessed only the effects ofpff on E2B-induced surges, either with or without previous pff immunization. In the saline-treated castrated monkeys (group 1) and the pff-immunized monkeys given concurrent pff (group 3), the E2B caused a familiar biphasic response, as shown in Figure 4. Three of four pffimmunized monkeys developed complete BIO-LH surge patterns (fastigium, 47.4 ± 8.0 fj.g/ml; n = 3) within 60 hours after estrogen treatment. In the other monkey, the LH surge was slightly delayed and was not fully completed within the study interval. Overall, the mean BIO-LH secretory responses of saline-treated versus pff-treated immunized monkeys were not different (P > 0.05) (Fig. 4). In contrast, all of the castrated monkeys that received pff but lacked prior pff exposure (nonimmunized; group 2) showed a significant decrease (P < 0.05) in the day 3 levels of BIO-LH but failed to demonstrate an E2B-induced BIO LH surge (Fig. 4). This difference in response to E2B was not due to pretreatment levels of BIO LH. All three groups of castrated monkeys had similar BIO-LH concentrations on day 1 (salinetreated, 31.3 ± 6.2 fj.g/ml; pff-nonimmunized, 31.5 ± 9.3 fj.g/ml; pff-immunized, 39.2 ± 8.0 fj.g/ml BIO-LH). As demonstrated on Ouchterlony diffusion gels (Fig. 5), the crude pff extract was apparently antigenic without an added adjuvant. The anti- GnAH! 60 i pff Treated GnAH GnAH GnAH ~ ~ t :!~;- : r i i! - I or- I I 316H : : : : pff Treated : ' i : m! 1 i/."i-\y\! i: I! i,, i!, i!!! NOON 2PM 4PM 6PM 8PM NOON 2PM 4PM 6PM BPM Figure 2 Composite patterns of BIO-LH in plasma from pff-treated ovariectomized monkeys receiving 50 ij.g GnRH at noon, 2:00, 4:00, and 6:00 P.M. on day 3. Florisil/charcoal-extracted pff administered at 8:00 A.M. (10 ml on day 1; 5 ml on days 2 and 3). Broken lines represent estimates of values above the detection limits of the assay. 110 Sopelak and Hodgen Gonadotropin surge inhibiting factor Fertility and Sterility

4 60 6O~----~sa~li-ne-o-rp~F~F~D~~ I-Ya~t~87A~M Saline Controls ~... pfftreated (non-immunized) NOON MID NOON- MID NOON MID NOON MID NOON MID NOON Figure 3 Plasma concentrations (mean ± SEM) of BIO-LH in control castrated monkeys (no pff) and pff-treated castrated monkeys that 'received 50 flglkg E2B on day 6. On day 3, these monkeys had received four injections of GnRH. Monkeys given pff received 10 ml Florisillcharcoal-extracted pff on day 1 and 5 ml pff on days 2 to 6. Control monkeys were significantly different (P < 0.05) from pff-treated females at all times after noon of day 2. serum from the four monkeys that received monthly injections' of pff showed precipitation bands on the Ouchterlony gels indicative of antigen-antibody complexes, while serum from saline-treated control monkeys was unreactive with the pff. DISCUSSION These findings illustrate that treatment of castrated monkeys with this regimen of a Florisill charcoal extract ofpff did not inhibit acute pituitary response (BIO-LH) to GnRH, although it did block E2B-induced LH surges. Further, chronic exposure to pff led to an apparent immune response that neutralized the capacity of pff to block estrogen-induced LH surges. Thus, in this regimen, pff differentially affected the LHsecreting response to GnRH and E2B challenges in long-term ovariectomized monkeys. We suggest that these differential effects of the pff extract may be due to distinct sites of action or receptors through which GnRH or E2B enhances BIO-LH secretion. In addition, more than one active pff constituent may be present. In primate studies, ovarian hyperstimulation, resulting from "pure" FSH or hmg therapy, attenuates the pituitary response to GnRH and blocks estrogen-positive feedback for the LH surge.1-4 Interestingly, we found that within 60 minutes following ovariectomy of hmg-treated monkeys, the LH response to a GnRH challenge was restored.12 We have suggested that in such ~ ~ ::t: 40..J 6 iii Saline or pff Daily at 8 AM SIIllne Controls instances, the hyperstimulated ovaries may be secreting follicular fluid constituents containing a substance(s) that can block the estrogeninduced LH surge13; further, this interpretation is consistent with the observations made here and elsewhere. 6 We have demonstrated in castrated monkeys that Florisillcharcoal,extracted pff can suppress BIO-LH surges induced by E2B. These results extend our earlier report of pff blockade of pituitary gonadotropin surges in intact monkeys given an estrogen challenge in the early follicular phase. 6 The collective indications of experiments directed toward studying the actions of pff on gonadotropin secretion in vivo and in vitro strongly implicate a direct action of pff on the pituitary. However, we feel one should remain open-minded to the potential effects ofpff on the hypothalamus. In the present study, because the same pff extract did not alter pituitary responsiveness to GnRH challenges, we are considering whether this pff factor(s) may block the estrogen-induced LH surge at the hypothalamic level, whereas GnRH surely acts directly on the pituitary. Previously, we showed that there is a gradual onset of refractoriness to pff-induced suppression of serum FSH in monkeys given repetitious injections of pff.14 Here, repeated injections of pff led to neutralization of its suppressive ef- pfft"'- ~... pff Non..fmmunized... pff Immunized 1:t,,,,,,,,,,,,,,,,,,,, NOON MID NOON MID NOON MID NOON MID NOON MID Figure 4 BIO-LH in control castrated monkeys (no pff) and pfftreated castrated monkeys (with and without pff immunizac tion) given E2B (50 flglkg; mean ± SEM). Saline treated and pff-immunized monkeys are statistically similar (P > 0.05). Nonimmunized pff-treated monkeys are significantly different from control monkeys or pff-immunized castrated monkeys at all times after 6:00 P.M. of day 2. Vol. 41, No.1, January 1984 Sopelak and Hodgen Gonadotropin surge inhibiting factor 111

5 NON-IMMUNIZED IMMUNIZED Figure 5 Ouchterlony diffusion gels. (A), Center well contains antigen (pff,. diluted 1:10) versus serum from non-pff-immunized castrated monkeys in wells 1 to 4; wells 5 and 6 contain buffer controls. (B), Center well contains pff (diluted 1:10); wells 1 to 4 contain. antiserum from pff -immunized monkeys; wells 5 and 6 contain serum from control monkeys injected with'pig serum. Notice the precipitation bands on B, indicative of antigen-antibody complexes. fects, thereby restoring E 2B-induced BIO-LH surges. From these findings we suggest an intrinsic antigenicityofpff, as indicated by neutralization of the factor(s) suppressing tonic FSH levels or estrogen-positive feedback for gonadotropin surges. Although the findings presented clearly distinguish effects of our pff extract on GnRH versus estrogen-induced LH secretion, others15 have reported that a charcoal extract of pff inhibited pituitary gonadotropin secretion in castrated monkeys given a GnRH challenge. Ultimately, this disparity may be accounted for by differences in the experimental design: the treatment regiment (dose and interval), the pff extract (Florisillcharcoal versus charcoal only), the GnRH dose, or the assay system employed (in vitro bioassay versus radioimmunoassay). We expect that additional studies will reconcile these differences. Conventionally, pff has been studied for its selective FSH-suppressing (inhibin/folliculostatin) activity.16~2o Because pff injections block estrogen-induced gonadotropin surges,6 we refer to this factor, with an activity distinct from the selective FSH suppression by inhibin/folliculostatin, as gonadotropin surge irihibiting factor (GnSIF). Studies need to be performed to determine whether human follicular fluid contains activity(ies) similar to that described here as a result ofpff treatment. We speculate that if a GnSIF also is found inhuman follicular fluid, this factor(s) may be secreted/exuded by monkey and human ovaries hyperstimulated with hmg or "pure" FSHbecause estrogen-induced LH surges are blocked.2,13 We acknowledge that the study described here using ovariectomized primates may have inherent differences from studies using intact primates; accordingly, we emphasize that prudence be used in extrapolating beyond the results presented. These findings encourage additional studies that describe further the physiologic and chemical nature of the inhibiting factor(s) of gonadal origin, whether imposed by injection of follicular fluid extracts or as may occur in the presence of gonadotropin-induced ovarian hyperstimulation.1-4, 6 Acknowledgments. The authors express their appreciation to Dr. Gabriel Bialy, Contraceptive Development Branch, NICHD, for the porcine follicular fluid and gonadotropinreleasing hormone, and to D.. L. Barber, A. W. Coleman, D. Greenawalt, A. Lynch, and B. Murphy for their technical assistance. Special thanks are extended to Miss L. Baldwin for typing the manuscript, and to the fellows of the Pregnancy Research Branch for their scientific comments.. REFERENCES 1. Garcia JE, Jones GS, Acosta AA, Wright G Jr: Human menopausal gonadotropinlhuman chorionic gonadotropin follicular maturation for oocyte aspiration: Phase I, Fertil Steril 39:167, Schenken RS, Hodgen GD: Follicle-stimulating hormone induced ovarian hyperstimulation in monkeys: blockade of the luteinizing hormone surge. J Clin Endocrinol Metab 57:50, Kreitmann 0, Lynch A, Nixon WE, Hodgen GD: Ovum collection, induced luteal dysfunction, in vitro fertilization, embryo development and low tubal ovum transfer in primates. In In Vitro Fertilization and Embryo Transfer, Edited by ESE Hafez, K Semm. New York, MTP Press Limited, 1980, p Goodman AL, Hodgen GD: Post partum patterns of circulating FSH, LH, prolactin, estradiol, and progesterone in nonsuckling cynomolgus monkeys. Steroids 31:731, dizerega GS, Turner CK, Stouffer RL, Anderson LD, Channing CP, Hodgen GD: Suppression of follicle-stimulating hormone-dependent folliculogenesis during the primate ovarian cycle. J Clin Endocrinol Metab 52:451, Hodgen G, Channing C, Anderson L, Gagliano P, Turner C, Stouffer R: On the regulation of FSH secretion in the primate hypothalamic-pituitary-ovarian axis. In Endocrinology, Proceedings of VI International Congress on Endocrinology, Edited by IA Cumming, JW Funder, FAO Mendelsohn. Canberra, Australian Academy of Science, 1980, p Sopelak VM, Lynch A, Williams RF, Hodgen GD: Maintenance of ovulatory menstrual cycles in chronically cannulated monkeys: a vest and mobile tether assembly. BioI Reprod 28:703, Van Damme MP, Robertson DM, Diczfalusy E: An improved in vitro bioassay method for measuring luteiniz- 112 Sopelak and Hodgen "Gonadotropin surge inhibiting factor Fertility and Sterility

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