Abstract. Introduction. RBMOnline - Vol 8. No Reproductive BioMedicine Online; on web 25 February 2004

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1 RBMOnline - Vol 8. No Reproductive BioMedicine Online; on web 25 February 2004 Article Induction of infertility in adult male bonnet monkeys by immunization with phage-expressed peptides of the extracellular domain of FSH receptor Professor AJ Rao obtained his PhD degree in 1971 from the Indian Institute of Science, Bangalore, and joined its faculty in 1979 after spending several years at the Hormone Research Laboratory, University of California in USA. His major scientific interests include regulation of endocrine function of placenta in primates, understanding the role of oestrogen in the modulation of epididymal function using monkey as a model, testing of compounds for anti-fertility activity (again, using monkey as a model), and understanding the reproductive biology of primates. Professor Rao has over 120 scientific publications to his name. Professor A Jagannadha Rao A Jagannadha Rao 1,4, SG Ramachandra 1, V Ramesh 1, L Couture 2, L Abdennebi 2, R Salesse 2, JJ Remy 2,3 1 Indian Institute of Science, Bangalore , India 2 Molecular and Cellular Biology, INRA Biotechnology, 78352, Jouy en Josas, France 3 Development Biology Institute of Marseille, NMDA Unit, UMR CNRS 6156, Marseille, Cedex 09, France 4 Correspondence: Tel: ; Fax: ; ajrao@biochem.iisc.ernet.in Abstract Active immunization of proven fertile adult male bonnet monkeys (Macaca radiata) with phage-expressed folliclestimulating hormone receptor (FSHR)-specific peptides from the extracellular domain resulted in a progressive drop in sperm count with all animals becoming azoospermic by day 100. However, serum testosterone concentrations were unaltered during the entire course of study and animals exhibited normal mating behaviour. Breeding studies with proven fertile female monkeys revealed that all the immunized males were infertile. Following interruption of immunization on day 225, sperm counts returned to normal with restoration of fertility. These results indicate that infertility can be induced in adult male monkeys by interfering with the action of FSH using specific peptides of the extracellular domain of FSHR as antigens, without the risk of producing cross-reacting antibodies to the other glycoprotein hormones. Keywords: antifertility agent, follicle stimulating hormone, libido, non-human primate, spermatogenesis, testosterone Introduction One of the important requirements for developing an acceptable male contraceptive is that it should not interfere with libido and secondary sexual characteristics. As testosterone is obligatory for maintenance of both, the choice of approaches that do not interfere with libido and secondary sexual characteristics is very limited. One such approach is the blockade of action of FSH, which has been shown to be essential for maintenance of spermatogenesis in the non-human primate. Studies by Moudgal et al. (1992) and Wickings and Nieschlag (1980), using bonnet monkeys and Rhesus monkeys respectively, have clearly established that immunization with ovine FSH results in decreased sperm counts and more importantly does not have any adverse effect on serum testosterone concentrations. Subsequently similar results were reported by immunization with the amino acid fragment of the FSH receptor (FSHR) (Moudgal et al., 1997). However, one of the problems with using the whole FSH or FSHβ as a candidate vaccine is the possibility of production of antibodies to the conserved regions of the related glycoprotein hormones, which may interfere with the function of these hormones. It is pertinent to note that FSH belongs to an evolutionary and structurally related family of glycoprotein hormones together with LH, thyroid-stimulating hormone (TSH) and human chorionic gonadotrophin (HCG). All the hormones are dimers consisting of a common alpha (α) subunit and a hormone-specific beta (β) subunit, which exhibits more than 40% homology among FSH/LH/TSH/HCG (Lapthorn et al., 1994; Fox et al., 2001). Similar problems may be encountered even when the FSHR is 385

2 386 used, as the receptors of all the above hormones belong to the large group of G protein-coupled receptors with 7- transmembrane regions, and the receptors also display the same degree of structural homology (Song et al., 2001). Furthermore, as demonstrated in the case of the TSH receptor (TSHR) in thyroid diseases, antireceptor antibodies can have either blocking or simulating activities depending on the epitope to which they have been generated (Orgiazzi et al., 2003). Thus the use of whole hormone or a large fragment of FSH or its receptor as an antigen may not be appropriate for generating highly specific antibodies to interfere with FSH action. Analysis of the amino acid sequences of FSH, LH and TSH receptors has revealed that there is a high degree of specificity, which is needed for membrane binding in the N-terminus region of the receptors. It has been demonstrated that antibodies against this region in the FSHR block FSHR activation in vitro (Couture et al., 1996; Remy et al., 1996; Nechamen and Dias, 2000). This paper reports the preliminary results of a study in which adult male bonnet monkeys were immunized with peptides corresponding to the FSHR decapeptides 18 27, and expressed individually on filamentous phages. Materials and methods Complementary oligonucleotide sequences encoding the decapeptides SKVTEIPSDL, SDLPRNAIEL and RNAIELRFVL of the human FSHR were ligated to the double-stranded DNA of the filamentous phage f88-4 (a kind gift from Professor G Smith, University of Missouri). Transformation of the E. coli strain MC1061 yielded recombinant tetracycline-resistant colonies. Overnight culture of single transformed colonies in NZY medium containing 40 µg/ml tetracycline yielded approximately 100 mg of phage particles. The presence of FSHR fragment was verified by sequencing hybrid coat protein P8 genes in every batch. The injected FSHR recombinant antigen used for immunization consisted of an equimolar mixture of the three recombinant phages. Animal experimentation Proven fertile 8 9-year-old monkeys of both sexes (Macaca radiata) from IISc colony were used in the study. Details on animal husbandry, housing and feeding have been described elsewhere (Rao et al., 1997). All experimental procedures including blood and semen collection were performed according to the protocols approved by the Indian Institute of Science Ethics committee for use of laboratory animals for biomedical research. Sperm analysis Semen samples were collected by electroejaculation once in every 15 days throughout the course of the study to monitor sperm counts. The instrument used in this study was designed and manufactured locally. Electroejaculation was carried out as described earlier (Ramesh et al., 1998). An electrode was applied at the base of the penile shaft and another electrode was applied with a clamp around the base of the glans penis. Intermittent charges of 20 V were delivered at a frequency of impulses per second for a period of 25 ms using a monophasic alternating current. Ejaculates were kept at 37 C throughout the period of examination and analysis of semen was generally completed within 90 min of collection. Sperm concentration was determined using a Neubauer hemocytometer following dilution of semen 1:200 with 0.9% NaCl, using the World Health Organization method (1992). Testicular tissue samples were collected by wedge biopsy technique under anaesthesia. Histological analysis of biopsy samples was carried out according to the standard procedure (Aravindan et al., 1990). Briefly, testicular biopsies obtained on day 200 were immediately fixed, dehydrated, paraffin waxembedded, sectioned serially and subjected to haematoxylin and eosin staining. Breeding studies Female monkeys were monitored every day for vaginal bleeding. Based on the spotting of blood, samples were collected from days 7 to 10 of the cycle for estimation of serum oestradiol-17β. Animals in which serum oestradiol-17β was more than 250 pg/ml on any day between 7 and 10 were cohabited with a control or immunized male monkey from day 10 to day 16 of the cycle. Each male (control and immunized) was exposed to at least three females during four menstrual cycles. Although immunizations were carried out throughout the year, breeding studies with the immunized males and the control animals, using proven fertile females, were only carried out during the breeding season. This is between July and February when the female animals exhibit regular menstrual cycles. Pregnancies were followed by ultrasonography and all the impregnated females delivered healthy babies. Details of active immunization Immunizations were performed by subcutaneous injection of 1 mg of phage mixture in Freund s complete adjuvant on day 1 followed by booster injection (without adjuvant) of 0.5 mg on day 25 and 0.1 mg on day 60. Thereafter subsequent boosters (0.1 mg) were given once every 30 days in normal saline. Monitoring FSHR antibody titre and serum testosterone radioimmunoassay For determination of immune response and testosterone concentrations, blood samples were collected by femoral vein puncture using sterile vacutainer tubes on specified days. Antibody titres were determined once every 60 days using a standard enzyme-linked immunosorbent assay (ELISA) procedure. Nunc immunoplate Polysorp (Nunclon, New York, USA) of 96 wells coated with 100 ng/100 µl of phage expressed recombinant FSHR peptides dissolved in phosphate-buffered saline, ph 7.4 was used for ELISA. The titres reported are for the antibodies against all three peptides used for the immunization and day 60 was the first day of antibody determination. Serum testosterone was determined at specific intervals by radioimmunoassay as described (Rao et al., 1998).

3 Results It can be seen from the results presented in Figure 1 that all four monkeys responded to the immunization with the FSHR peptides expressed on phage particles. The values expressed are over and above the titre detected in the animals injected with the phage particles. It is evident that the response was variable, and following a booster around day 360, higher titres could be detected in all four animals. The results presented in Figure 2 indicate that the serum testosterone concentration was unaffected during the entire course of the study. All the animals exhibited the typical nocturnal surge of serum testosterone, a characteristic feature observed in adult bonnet monkeys maintained under regulated light conditions and captivity (Mukku et al., 1981). Analysis of sperm counts revealed that all the monkeys had sperm counts in the range of per ejaculate before starting the study (Figure 3). Sperm counts started decreasing by day 30 of immunization and the decrease was significant by day 60 (P < 0.001). All the animals were azoospermic by day 105 and the sperm counts remained low in the range of per ejaculate between days 150 and 225 (Figure 3). Immunization was carried out between days 0 and 225 and breeding studies with proven fertile females were carried out between days 90 and 225; as stated above, during this period the animals exhibited azoospermia to oligozoospermia. Results of the breeding studies revealed that while nine pregnancies were recorded in the control group (animals injected with only phage particles), no pregnancies were recorded in the FSHR peptide-immunized group. Figure 1. Antibody titre (1:1000) in adult male bonnet monkeys (n = 4) immunized against phageexpressed human FSH receptor peptide. Figure 2. Serum testosterone concentrations in adult male bonnet monkeys (n = 4) immunized against phage-expressed human FSH receptor peptide. Values are mean ± SEM. 387

4 Table 1. Results of fertility study carried out in monkeys immunized against phage-expressed human FSH receptor peptides from days and of immunization. Number of females exposed Group I a 12 9 (n = 4) Group II b 12 0 (n = 4) Group II c 12 4 (n = 4) Number of females that conceived a Immunized with phage alone. b Immunized against phage-expressed human FSH receptor peptide ( days). c Data collected during recovery period ( days). Interestingly, following interruption of immunization on day 225, the sperm counts started increasing gradually and reached normal values by day 300. Breeding studies carried out during the recovery period (days ) revealed that all the immunized males were able to impregnate the females, indicating a complete recovery of fertility (Table 1). It was observed that variable periods of immunization were needed to achieve a total inhibition of sperm production. However, the decrease in sperm count and induction of infertility in the monkeys were not dependent upon the variation observed in the specific antibody titre (Figure 4). Thus by day 105, all the animals were azoospermic although the antibody titres varied from a value of 0.2 to 0.6 on day 120 of immunization. Although regression analysis demonstrates a linear relationship between antibody titre and sperm count values (Figure 5), analysis of the antibody titres and sperm counts throughout the course of the study reveals that at certain time points there was no direct correlation in fact on certain days when the antibody titre was very high, the sperm counts were also high when they should have been low. It is possible that induction of infertility is directly related to the proportion of antibodies generated that are capable of binding to FSHR thereby blocking FSH action. Although not included in the present paper, video recordings of mating studies of immunized animals revealed that all animals exhibited instantaneous mounting and normal sexual behaviour. Histological analysis of the testicular biopsies collected from all the immunized and control animals on day 200 of immunization revealed a distorted tubular architecture with a reduction in the size of tubules and number of spermatogonia in the immunized animals (34 ± 2.9; n = 3; Figure 6a c) when compared with controls (73 ± 1; n = 2; Figure 6d). This indicates that the action of FSH was blocked in these animals. Discussion The importance of FSH for initiation of spermatogenesis in primates is well documented. Although based on the results obtained with recent studies using knockout models of FSH and FSHR knockout (FRKO) mice, it has been suggested that FSH is not needed for spermatogenesis in rodents (Kumar et al., 1997; Tapanainen et al., 1997; Dierich et al., 1998; Lindstedt et al., 1998). Careful analysis of the data obtained with rodent models, and of clinical data reveals that the spermatogenesis in these cases is not normal and that for quantitatively normal spermatogenesis FSH is absolutely essential (Strehler et al., 1997). The results of the present study clearly establish that FSH is needed for normal spermatogenesis and interference with FSH action results in infertility. Furthermore it is also evident that a small portion of the specific region of the extracellular domain of FSHR is adequate for eliciting antibodies that are capable of interfering with the interaction of FSH with its cognate receptor. Moreover, in the period when breeding studies were carried out, the sperm counts ranged from azoospermia to extreme oligozoospermia (< per ejaculate). It is possible that the quality of the remaining spermatozoa was drastically affected resulting in infertility. In addition, the ovulatory nature of each of the cycles during which immunized or control monkeys were exposed to females was ascertained by 388 Figure 3. Sperm count in adult male bonnet monkeys (n = 4) immunized against phage-expressed human FSH receptor peptide. Values are mean ± SEM.

5 Figure 4. Sperm count and antibody titre of adult male bonnet monkeys (n = 4) immunized against phage-expressed human FSH receptor peptide. Values are mean ± SEM. Figure 5. Correlation between antibody titres and sperm count. The correlation curve, obtained by using a Statview 512+ program (Brain Power Inc., Miami, FL, USA) was obtained by comparing antibody titres (OD 450 nm) and sperm counts ( 10 6 /ejaculate) in the FSHR-vaccinated animals. Antibody titres were determined by ELISA against the mixture of injected phages and calculated as OD 450 nm differences between immune and non-immune sera at a 1:1000 dilution. measuring serum oestradiol-17β between days 7 and 10 in each of the females. Estimation of serum oestradiol-17β was done retrospectively and only those cycles in which it was above 250 pg/ml were included for calculating the number of cycles during which the female monkeys were exposed to the males. Although earlier studies have established that infertility can be induced in non-human primates by interfering with the action of FSH, the present study clearly establishes for the first time that the action of FSH can be blocked by generating antibodies to a small specific region of the FSHR. Thus it appears that the small peptides from the FSHR are quite efficient in generating neutralizing antibodies, although a recent theory proposed by Pieczenik (2003) suggests that peptides are less efficient. In contrast to the classical theory on diversity of antibodies, which assumes that the number of possible interacting antibodies is infinite, the new theory suggests that this number is limited by the fact that the binding specificity of monoclonal antibodies of four to six amino acids limits the total number of antibody-binding peptides. Thus, based on this theory, the approach of using peptides as antigens appears to be less efficient. However, it is pertinent to note that infertility was induced in adult female bonnet monkeys by immunizing against a synthetic peptide corresponding to residues of zona glycoprotein C-conjugated diphtheria toxoid (Kaul et al., 2001). Also the synthetic peptide showed better immunocontraceptive potential as an antigen compared to the whole zona pellucida protein. It is possible to mass produce the phage particles to express specific peptides or further reduce the size of the peptides needed for blocking the FSH action. This study also provides an opportunity to develop approaches for investigating the interaction between the FSHR and its ligand so that subsequently a small non-peptide molecule could be developed to block the action of FSH instead of relying on immunization with FSHR or its peptides. As indicated earlier, the use of small specific regions of the FSHR peptides eliminates the possibility of generating antibodies to other conserved regions of the G-protein coupled receptors (GPCR), which are likely to interfere with the action of other related hormones. In addition, the sparse distribution of the targeted antigen, FSHR, which is localized only in Sertoli cells, provides a better chance of it being an efficient antigen at lower antibody titres even in lowresponding subjects. Comparison of haematological and 389

6 a b c d Figure 6. Histological sections (stained with haematoxylin and eosin) of testes of adult male bonnet monkeys immunized against phage-expressed human FSH receptor peptide (a, monkey no. 618; b, monkey no. 567; c, monkey no. 590) and control monkeys immunized with phage alone (d). Testicular biopsies obtained on day 200 were immediately fixed, dehydrated, paraffin wax-embedded, sectioned serially and subjected to haematoxylin and eosin staining. Original magnification is biochemical analysis of serum of the control and FSHRimmunized animals revealed that there were no adverse effects in any of the animals during the entire period of immunization (data not presented). The results of the present study clearly establish that infertility in non-human primates can be induced by employing a specific region of the FSHR without recourse to the use of whole hormone or a large fragment of FSHR as used in earlier studies. Although several methods of contraception are available, there is still a largely unmet need for new and improved methods that are easier to use, are under the user s control, have fewer side-effects and respond to the needs of different groups of users including men. As discussed earlier, one of the major problems in developing a usable male contraceptive is that it should not interfere with libido. In this investigation small peptides from FSHR were used for generating antibodies, which blocked the action of FSH, and induced infertility in male bonnet monkeys without interfering with libido or secondary sexual characteristics. These results are significant and important. Acknowledgements The authors thank Professor George Smith for the kind gift of f88-4 vector, Denise Grebert for excellent technical assistance and Dr V Machelon for helpful discussion. AJ Rao thanks Mellon Foundation and CONRAD, USA, Department of Biotechnology, Indian Council of Medical Research, the Government of India and the Indian Institute of Science, Bangalore for financial assistance. References Aravindan GR, Ravindranath N, Gopalakrishnan K et al DNA flowcytometric analysis of testicular germ cell populations of the bonnet monkey (Macaca radiata) as a function of sexual maturity. Journal of Reproduction and Fertility 89, Couture L, Naharisoa H, Grebert D et al Peptide and immunochemical mapping of the ectodomain of the porcine LH receptor. Journal of Molecular Endocrinology 16, D Arcangues C 2001 Family planning needs: new opportunities, emergency contraception and other new technologies. Reproductive BioMedicine Online 3, Dierich A, Sairam MR, Monaco L et al Impairing follicle-

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