Active immunization with follicle-stimulating hormone for fertility control: a 41J2-year study in male rhesus monkeys

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1 FERTILITY AND STERIIJTY Copyright ~ 1983 The American Fertility Society Vol. 40, No. I, July 1983 Printed in U.8A. Active immunization with follicle-stimulating hormone for fertility control: a 41J2-year study in male rhesus monkeys Banavar R. Srinath, B.Sc. * E. Jean Wickings, Dr.Rer.Med.* Christian Witting, Prof.Dr.Med.t Eberhard Nieschlag, Prof.Dr.Med. *; Max Planck Clinical Research Unit for Reproductive Medicine and Institute of Pathology, University of Munster, Munster, Federal Republic of Germany Active immunization of four adult rhesus monkeys with highly purified ovine follicle-stimulating hormone (FSH) over 4% years resulted in the production of specific FSH antibodies. While luteinizing hormone and testosterone secretion were not affected, sperm counts were in most instances reduced to or below the lower normal range. On a few occasions, azoospermia or high sperm counts were observed. Although the antibodies produced neutralized the biologic activity of FSH throughout, spermatogenesis gradually returned, as evidenced by testicular histologic characteristics. However, the diameter of the seminiferous tubules remained decreased, and the germinal epithelium was depleted. No adverse side effects could be demonstrated in the immunized animals, e.g., immune complexes in either serum or tissues and resultant tissue damage. These results show that although active immunization with FSH may not result in an effective method of male fertility control, long-term immunization against a circulating hormone may not result in deleterious side effects. Fertil Steril40:110, 1983 In 1977, the World Health Organization Task Force on Regulation of Male Fertility initiated studies to clarify the role of follicle-stimulating hormone (FSH) in nonhuman primates. The main aim was to provide information on whether FSH plays a role in spermatogenesis in the mature Received January 3,1983; revised and accepted March 17, *Max Planck Clinical Research Unit for Reproductive Medicine, University Women's Hospital. tlnstitute of Pathology, University of Munster. treprint requests: Professor Dr. E. Nieschlag, Max Planck Clinical Research Unit for Reproductive Medicine, Steinfurterstrasse 107, Miinster, Federal Republic of Germany. 110 Srinath et ai. FSH immunization for fertility control primate and also whether selective neutralization of FSH could lead to the development of a method for the regulation of male fertility. Short-term studies in various macaque species have shown that the neutralization of FSH leads to the suppression of spermatogenesis in the mature animal. 1 3 This study was designed to investigate whether FSH can be neutralized and, hence, whether spermatogenesis can be suppressed over prolonged periods; in this instance, 4% years. In addition to the effects on spermatogenesis, specific attention was paid to any immunologic side effects in the long-term immunized monkeys. The results of the first 2 years of immunization have already been published,4 but they are briefly included here for completeness. Fertility and Sterility

2 MATERIALS AND METHODS EXPERIMENTAL DESIGN Four sexually mature rhesus monkeys were actively immunized with highly purified ovine FSH in November Four weight-matched animals served as controls for the first 18 months. The ability of the antibodies to neutralize FSH was checked in vitro and in vivo using human and rhesus FSH, and antibody titer and specificity were also monitored. Serum luteinizing hormone (LH) and testosterone (T) levels were measured by in vitro bioassay5 and radioimmunoassay6 without chromatography. Sperm counts, motility, and morphologic features were determined in the ejaculates collected by penile electrostimulation, and testicular length and width were measured for computation of testicular volumes. 7 The frequency of spontaneous ejaculation was determined by counting the number of ejaculatory plugs found under the monkeys' cages each morning. These parameters were monitored at approximately biweekly intervals over the following 4% years until the animals were sacrificed in May ANIMALS The monkeys were housed under standard conditions in individual cages in a temperature-, humidity-, and light-controlled room. The staple diet was a standard monkey pellet food, which was supplemented daily with fresh fruit and vegetables. Water was also available ad libitum. The animals were totally isolated from female monkeys throughout the study. All handling procedures were carried out under ketamine anesthesia (Vetalar, Parke-Davis, Freiburg, FRG), 8 to 12 mg/kg body weight, given intramuscularly. ANTIBODY CHARACTERIZATION The highly purified ovine FSH used as antigen for immunization and the primary intradermal immunization procedure using complete Freund's adjuvant have already been describ.ed in detail.1, 4 Booster injections were given at regular intervals, using - 50 fj.g ovine FSH in incomplete Freund's adjuvant injected subcutaneously and intramuscularly. The last booster immunization was given in November Antibody titers were measured in all the blood samples collected.1 The in vivo neutralizing ca- Vol. 40, No.1, July 1983 pacity of individual antisera (February to May 1978) was initially checked in the Steelman-Pohley FSH bioassay8 against a rhesus pituitary preparation (LER ) and human FSH (Pergonal, Serono Pharmazeutica, Freiburg, FRG). Antisera tested after 4 years of immunization (January 1982) were checked only against human FSH in the bioassay. The test rats received two injections of antisera (5 fj.1) with the first and fourth injections of FSH. Cross-reactivity of the antisera toward LH was monitored radioimmunologically with 125I_Ia_ beled human LH (20,000 CPM LER 960). Iodinelabeled ovine LH (LER 1056-C-2) and rat LH (LH-S-l) were also tested. Serum at an end dilution of 1:5000 was incubated with 20,000 CPM of each iodinated hormone, as described for the determination of antibody titer.1 All iodinations were performed by the chloramine T method. 9 HISTOLOGIC INVESTIGATIONS Testicular biopsies were performed under sterile conditions on three occasions during the first year of immunization. 4 Tissue was carefully removed through an incision in the tunica albuginea. Approximately 50 to 100 mg tissue were removed on each occasion, and alternate testes were used for successive biopsies. When the animals were sacrificed at the end of the study (May 1982), the testes were removed. All tissues were fixed in Bouin's solution, imbedded in paraplast, and cut into 5-fJ.m slices. These were stained with periodic acid-schifflhematoxylin. In addition to evaluating the histologic features under the light microscope, the diameters of the seminiferous tubules were evaluated histometrically from 50 measurements per monkey. For quantitation, only seminiferous tubule cross-sections containing stage VI cellular associations were evaluated. IDENTIFICATION OF SOLUBLE CIRCULATING IMMUNE COMPLEXES Chromatographic separation of immune complexes was carried out by the method of Kilpatrick and Virella. lo The "V-globulin protein fraction was first precipitated out of the serum using polyethylene glycol 6000, and any immune complexes were isolated by chromatography on Sephacryl S200 Superfine and then on Protein-A Sepharose (Pharmacia, Freiburg, FRG). As a control, serum from a vasectomized rhesus monkey, Srinath et al. FSH immunization for fertility control 111

3 l YlOOK.. " V50K V25K V5K ( mean. S.f'. ) NDJFMAMJJASONDJFMAMJJASONDJFMAMJJASONDJFMAMJJASONDJFMAM Figure 1 Anti-FSH titers in sera from four adult male rhesus monkeys actively immunized with ovine FSH as measured over 4V2 years. The small arrows indicate booster injections following primary immunization. known to contain immune complexes, was processed in an identical manner. IDENTIFICATION OF IMMUNE COMPLEXES IN TISSUES BY IMMUNOFLUORESCENCE Identification of immune complexes in tissues by immunofluorescence was performed as described by Witting et al. ll The tissues examined included kidney, aorta, heart, lung, brain, liver, spleen, pancreas, eye, muscle, testis, prostate, and vas deferens. Cryostat slices of tissues were incubated with fluorochrome-labeled polyvalent goat anti-monkey ),-globulin and evaluated under the fluorescence microscope. The specificity of the fluorescence reaction was checked by incubating slices of tissues with fluorescein isothiocyanate-coupled anti-monkey albumin serum. STATISTICAL EVALUATION The Student's unpaired t-test was employed to analyze the endocrine data, testicular volumes, and histometric measurements. The chi-square test was employed to compare the frequencies of spontaneous ejaculation. RESULTS No deleterious side effects of immunization were noted, with the animals remaining apparently healthy and active. None of the monkeys showed any general adverse reactions to immunization. Body weight tended to increase throughout the study. ANTIBODY CHARACTERISTICS The four actively immunized monkeys showed detectable antibody titers 6 weeks after primary 112 Srinath et al. FSH immunization for fertility control immunization. Frequent booster immunizations were able to maintain high titers of antibodies until the end of the study. The mean of the antibody titers was 1:90,000 ± 1:27,000 (mean ± standard error [SE]) (Fig. 1). The neutralizing capacity of the individual antiserum in the Steelman-Pohley bioassay was maintained in the range of 0.6 to 16 IV human FSH per 10 fl.l of antiserum, as previously described. 4 In a pool of serum obtained from all immunized monkeys between January and July 1978, no cross-reactivity toward human LH could be demonstrated. 1 However, in vitro radioimmunologi(; studies showed high specific binding of iodinated ovine LH (20% to 60%) and rat LH (20% to 60%) to individual antisera, but with very low binding of I25I_human LH. The binding of human LH was below 20% and was usually indistinguishable from the nonspecific binding. TESTICULAR VOLUMES Testicular volumes in the control monkeys showed characteristic seasonal variations ranging from 34 ± 8 ml (mean ± SE) in season to 13 ± 2 ml (mean ± SE) out of season. By the time antibody titers were detected in the immunized monkeys, the testicular volumes were significantly below the control values (P < ) and continued to decrease until they reached out-ofseason values in January 1978 (Fig. 2). Following this dramatic decrease, testicular sizes increased gradually throughout the remainder of the observation period but did not reach normal in-season values thereafter. The seasonal variations evident in the controls were no longer seen in the immunized monkeys. Fertility and Sterility

4 50 40., 30 E :2 0 > E as.. :2 10 U II)., ~ 200 T r T.L~ T l/ ~ ~." ' -, : ~.-O T ~/ " p-- N 0 J F M A M J J A SON 0 J F M A M J J A SON 0 J F M A M J J A SON 0 J F M A M J J A 5 '-,: 150 1: E..I... E :2... II t/) ~ E , C.... o., o _ II) II) 0 20! ~ E :2...., t/) Figure 2 Testicular volumes and serum LH and T concentrations in four normal (0) and four actively immunized (e) monkeys. Observations in control animals show typical seasonal variations in all parameters. Measurements were discontinued after April 1979, and the dotted line represents a repetition of control data for previous years (mean ± SE). Vol. 40, No.1, July 1983 Srinath et al. FSH immunization for fertility control 113

5 N. R. = not recorded FSH immunized monkeys (n = 4) o Control monkeys (n = 16) N 1977 Figure 3 Frequency of spontaneous ejaculations in normal and FSH immunized monkeys as estimated from the ejacula tory plugs found under the monkeys' cages each morn ing. EJACULATORY BEHAVIOR The frequencies of spontaneous ejaculation were similar in both the control and immunized groups of monkeys, and may even have been slightly increased in the latter group (Fig. 3). The response to electrostimulation was also not different between the two groups. SEMINAL PARAMETERS Ejaculate weights did not change significantly throughout the immunization period (preimmunization,1.38 ± 0.84 gm [mean ± standard deviation]; postimmunization, first year, 0.94 ± 0.79 gm; second year, 1.25 ± 0.82 gm; third year, 1.31 ± 0.89 gm; and fourth year, 2.33 ± 2.12 gm). Seminal parameters are depicted in Figure 4.7 In most instances over the 41/z-year period, sperm counts were in or below the lower normal range of 110 million sperm per ejaculate. Azoospermia was only seen occasionally, in monkeys 1 and 3. Occasionally, high sperm counts were also observed. For the first 2 years of immunization, the number of normal forms was consistently reduced, thereafter returning to the normal range. Motility, although subject to some fluctuations, did not appear to be grossly affected. One monkey (monkey 3) showed azoospermia on four occasions during the last year of immunization, and on other occasions there were so few sperm in the ejaculates that it was very difficult. to quantitate motility and normal forms. The normal seasonal pattern of high sperm counts in season and the failure to respond to electro stimulation during the out-of-season period also seemed to be affected in this same monkey, because the highest sperm counts were obtained in August and September, whereafter ejaculate quality deteriorated. ENDOCRINE PARAMETERS Serum levels of biologically active LH were not different from control values during the first 18 months of immunization but thereafter tended to be lower than control values (Fig. 2). Serum T concentrations in the immunized monkeys, following an initial increase over the control values, did not differ thereafter and still showed typical seasonal variations (Fig. 2). TESTICULAR HISTOLOGY Spermatogenesis was severely impaired during the first year of immunization, and the tubular diameter was 108 ± 2 j.lm, as compared with control values of 182 ± 4 j.lm, in December Some tubules contained only Sertoli cells and spermatogonia. Thickening of the basal membrane was also evident. By the end of the study, spermatogenesis had been reestablished, although only qualitatively. Quantitatively, the cell numbers per cross-section, in particular, the numbers of spermatids, were reduced. The tubular diameter had increased to 134 ± 15 j.lm but was still below normal values (Fig. 5). The Leydig cells appeared normal, although it was not possible to make a detailed examination using the periodic acid-schifflhematoxylin-stained sections. This staining was chosen because it is particularly suitable for the stages of the cycle of the seminiferous epithelium. DEMONSTRATION OF IMMUNE COMPLEXES Figure 6 shows the elution patterns of immune complexes isolated from the vasectomized monkey serum where circulating immune complexes were known to be present. No such high-molecular-weight complexes could be identified in any of 114 Srinath et al. FSH immunization for fertility control Fertility and Sterility

6 ~lr ~ TOTAL SPERM I EJACULATE I tmwn~mnnc~~~~an~~~ t%i~ , 100 NORMAllY FORMED SPERM Figure 4 Sperm counts (total sperm per ejaculate) and the percentage of motile and normally formed sperm in ejaculates obtained from adult rhesus monkeys actively immunized with ovine FSH in November 1977 as compared with seminal parameters mea sured in 37 ejaculates from normal in season monkeys (shaded area).7 The numbers 1 to 4 identify the individual animals. the FSH antisera tested. No precipitated immune complexes could be demonstrated in any of the tissues examined (kidney, heart, lung, brain, liver, aorta, eye, spleen, pancreas, muscle, testis, prostate, and vas deferens). All these organs were also macroscopically and histologically normal. DISCUSSION We have previously demonstrated that spermatogenesis in the mature rhesus monkey can be specifically suppressed by both active and passive immunization against FSH.1, 4 This is in direct contrast to the effects resulting from the neutralization of FSH in adult rats. Administration of FSH antiserum for 14 or 30 days had no effect on the light- or electron-microscopic morphologic characteristics of the testis, and the immunized rats showed no signs of impaired libido or fertility.12 Extending the initial observations made in the actively immunized monkeys to a period of 41/2 years revealed that spermatogenesis had recovered to such an extent that the animals could no longer be considered infertile. Sperm counts were generally in or below the lower normal range, but sperm motility and normal forms were not adversely affected. After the initial drop, testicular volumes gradually increased; and although spermatogenesis remained reduced, the tubular diameters had also increased at the end of the study. Several reasons for this recovery of spermatogenesis may be discussed. FSH neutralizing capacity in vivo was as high at the end of the study as in the first 6 months. Radioimmunologically determined titers remained high throughout the study, and from the correlation demonstrable between antibody titer in vitro and neutralizing capacity4 in vivo, it can be suggested that the high FSH neutralizing capacity was maintained over the entire period. Unfortunately, the lack of a pure rhesus pituitary FSH preparation precluded quantitating antibody affinity and binding capacity. Ovine FSH was used as antigen because this was the purest FSH preparation available to us at the commencement of the study. However, we were interested primarily in the behavior of the circulating antibodies to rhesus FSH, and in view of the fact that no rhesus preparation of comparable purity is available, human FSH was used for checking neutralizing capacities and cross-reactivity. It was chosen on a phylogenetic basis, assuming that rhesus and human hormones show more similarities than rhesus and ovine hormones. Hence, the possibility that changes in antibody characteristics had occurred during prolonged immunization with repeated booster injections cannot be excluded. Another possible explanation is that FSH is not the only factor required for the maintenance of spermatogenesis in primates. Endocrine testicular function in these monkeys was intact throughout the study, as judged by the serum T levels and the frequency of spontaneous ejaculation. Other studies in FSH-immunized monkeys have shown that sperm counts could be suppressed but that azoospermia could not be consistently produced.2, 3 In men in whom spermato- Vol. 40, No. I, July 1983 Srinath et al. FSH immunization for fertility control 115

7 Figure 5 Histologic features of testes from (a) a normal rhesus monkey in season, with fully developed germinal epithelium; (b) monkey 3 12 months after primary immunization, showing narrow tubules, lumina full of degenerating spermatogenic elements, and spermatogenic arrest; (c) monkey 3 at the end of the study, showing only partial regeneration of the seminiferous epithelium and reduced tubule diameter (periodic acid-schifflhematoxylin, original magnification, x 300). genesis had been suppressed by high-dose T treatment, sperm counts could be increased by the administration of human chorionic gonadotropin alone. 13 In rhesus monkeys where hypophyseal stalk section has been performed,14 spermatogenesis can in fact be stimulated to a certain degree by T alone. In neither of these two studies, however, were sperm counts restored to normal. In the present study, the neutralization of FSH initially caused dramatic effects on exocrine testicular function; but with continued immunization and intact endocrine activity, other factors, such as intratesticular T may become more effective in supporting spermatogenesis. The other important feature of this study was to observe the side effects oflong-term immunization of the animals using complete Freund's adjuvant. One such possible effect would be the formation of immune complexes potentially leading to tissue damage such as nephritis or atherosclerosis. No soluble immune complexes could be demonstrated in any of the serum samples tested over the entire immunization period. Neither could tissue immune complexes be identified after the animals were sacrificed. In this context, it is worthwhile to mention that no side effects were reported in any of the 63 women actively immunized with a J3-human chorionic gonadotropintetanus toxoid vaccine. 15 In addition, Wass and co-workers l6 found that spontaneously occurring 116 Srinath et ai. FSH immunization for fertility control circulating antibodies to human chorionic gonadotropinllh did not produce any adverse reaction, indicating that antibodies to a humoral body constituent need not necessarily be hazardous. In summary, these studies show that long-term neutralization of FSH suppresses spermatogenesis to a certain extent, but not to the required level of azoospermia if active immunization II ~ak Slip I fgdtklltlitionj 30 Sample t.icir'w~ strum from NJAIPEG p~d CGl.tmn Stphac'Yl- S 21)0 Eluant Tns tti Mer 20 Slap IIIAffinty. o.malallqftfl ~. I PlGkpoolcd fratbclls frum GdfIrWon Column PIIIkIn A Septaua Eluant I f'hmptii:dt adkr II 1M Aatlt acd 45 Volirreol froctloos 1m! Figure 6 Chromatograph of serum from a vasectomized rhesus monkey, showing the elution pattern of soluble immune complexes. No such high-molecular-weight complexes were found in sera from FSH-immunized monkeys at any time. Fertility and Sterility

8 against FSH should be considered a method of male fertility control. One positive aspect of the study was the failure to identify any adverse side effects, even though the virulent Freund's adjuvant was used for primary immunization. Acknowledgments. The initial phases of the study were supported by the World Health Organization Human Reproduction Programme, Geneva, Switzerland. The highly purified ovine FSH preparation (S 1554 BR) was generously supplied by Dr. M. R. Sairam, Montreal, Quebec, Canada. The rhesus pituitary preparation (LER ) was the gift of Professor L. E. Reichert, Atlanta, Georgia, to whom we are very grateful. The 1st IRP Bioassay Standard for LH and FSH was donated by the Institute for Biological Standards and Control, London, England, and the other pituitary preparations were donated by the NIAMDD, Baltimore, Maryland. The vasectomized rhesus monkey serum was generously provided by Dr. N. J. Alexander, Beaverton, Oregon. REFERENCES 1. Wickings EJ, Usadel KH, Dathe G, Nieschlag E: The role of FSH in testicular function of the mature rhesus monkey. Acta Endocrinol (Copenh) 95:117, Murty GSRC, Sheela Rani CS, Moudgal NR, Prasad MR: Effect of passive immunization with specific antiserum to FSH on the spermatogenic process and fertility of adult male bonnet monkeys (M. radiata). J Reprod Fertil Suppl 26:157, Madhwa Raj HGM, Sairam MR, Dym M, Kotite N, French FS, Sloan C, Chen DR: Effect of immunization against follicle-stimulating hormone on spermatogenesis in the rat and the monkey. In Non-human Primate Models for the Study of Human Reproduction, Edited by TC Anand Kumar. Basel, S. Karger, 1980, p Wickings EJ, Nieschlag E: Suppression of spermatogenesis over two years in rhesus monkeys actively immunized with follicle-stimulating hormone. Fertil Steril 34:269, Nieschlag E, Loriaux DL: Radioimmunoassay for plasma testosterone. Z Klin Chern Biochem 10:164, Wickings EJ, Qazi MH, Nieschlag E: Determination of biologically active LH in the serum of male rhesus monkeys (M. mulatta). J Reprod Fertil 57:497, Wickings EJ, Nieschlag E: Seasonality in endocrine and exocrine testicular function of the adult rhesus monkey (Macaca mulatta) maintained in a controlled laboratory environment. Int J Androl 3:87, Steelman SL, Pohley FM: Assay of the follicle-stimulating hormone based on the augmentation with human chorionic gonadotropin. Endocrinology 53:604, Hunter WM, Greenwood FC: Preparation of labelled growth hormone of high specific activity. Nature 189:495, Kilpatrick JM, Virella G: Isolation and charetherization of soluble insulin and anti insulin immunecomplexes formed in vitro and in vivo in sera from patients with diabetes mellitus. Clin Exp Immunol 40:445, Witting C, Wickings EJ, Nieschlag E: Incidence of immune complex nephritis following active immunization with a testosterone-3-bsa conjugate or BSA alone. Acta Endocrinol (Copenh) 90:562, Dym M, Raj GGM, Lin YC, Chemes HE, Kotite NJ, Nayfeh SN, French FS: Is FSH required for maintenance of spermatogenesis in adult rats. J Reprod Fertil Suppl 26:175, Bremner WJ, Matsumoto AM, Sussman AM, Paulsen CA: Follicle-stimulating hormone and human spermatogenesis. J Clin Invest 68:1044, Marshall GR, Wickings EJ, Liidecke DK, Nieschlag E: Restoration of spermatogenesis in stalk-sectioned rhesus monkeys by testosterone alone. J Clin Endocrinol Metab. In press, July, Talwar GP: Vaccines based on the J3-subunit of HCG. In Immunological Aspects of Reproduction and Fertility Control, Edited by JP Hearn. London, MTP Press, 1980, p Wass M, McCann K, Bagshaw KD: Isolation of antibodies to hcg/lh from human sera. Nature 271:368, 1978 Vol. 40, No.1, July 1983 Srinath et a1. FSH immunization for fertility control 117