Immunomodulation of rodent male fertility by vaccination against the luteinizing hormone receptor

ELSEVIER

Livestock Production

Science 42 ( 1995) 207-2

I1

Immunomodulation of rodent male fertility by vaccination against the luteinizing hormone receptor Jean-Jacques Remy”, Laurence Couture, Roland Salesse Unite’d’lng&ierie des Protknes, lnstitut National de la Recherche Agronomique, 78352 Jouy-en-Josas, France

Abstract Luteinizing hormone (LH) controls, through interaction with its specific receptor, the steroidogenic activity of the testicular Leydig cells. The possibility of antagonizing LH activities by means of immunization against its receptor was investigated. Immunization of prepubertal male rodents against the N-terminal extracellular binding domain of the porcine LH receptor impaired fertility. Male rats were less affected than male mice. Circulating maternal anti-LH receptor antibodies supressed fertility of the young rats to the same extent as did active immunization. In view of designing a more efficient and specific antagonist of the LH receptor, potentially usable as an immunocastration tool, a peptide involved in hormone-receptor interaction (LH receptor peptide 20-36) was used as the antigen. Immunization of prepubertal male mice against the peptide qualitatively reproduced the effects of immunization against the whole binding domain of the receptor: antifertility was mostly due to sterilization and/or delayed initiation of fertility, rather than to impaired fecundity. Keywords: lmmunocontraception

-

male; Immunocastration;

Gonadotrophin

1. Introduction In modulating male fertility, two strategies have been developed. The first one consists of immunizing against sperm antigens in order to obtain infertility (Primakoff et al., 1988; Naz, 1992). The second represents the endocrine approach, and acts on the steroidogenic gonadotrophins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), through immunization against them, or through inhibition of their secretion from the pituitary (Mougdal et al., 1988; Weinbauer and Nieschlag, 1992). Both strategies emanated from clinical observations where infertility in man, among other causes, can result from autoimmunization against sperm antigens or from hypogonado* Corresponding

author.

0301-6226/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved .SSD/O301-6226(95)00022-4

receptor

trophic hypogonadism, the latter being successfully reversed by hormonal treatments. In males, LH and FSH act through their specific membrane receptors, present on Leydig and Sertoli testicular cells, respectively. Theoretically, blocking LH action would result in an inhibition of androgens production by Leydig cells which therefore would impair spermatogenesis, while blocking FSH action on Sertoli cells would lead to oligo or azoospermia without affecting secretion of androgens. Recent molecular cloning of gonadotrophin receptor cDNA (reviewed by Salesse and Gamier, 1994) rendered possible to explore a new way of modulating gonadotrophin activities through the targetting of their receptors. Gonadotrophin receptors belong to the superfamily of G-protein coupled receptors with seven transmembrane segments, and exhibit large (half of the molecule) extracellular N-

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terminal domains able to bind hormones. Luteinizing hormone plays an essential role in the developement of the male genital tractus, in the initiation of spermatogenesis (except in rat, where FSH is essential), male characteristics and male behaviour. Blocking LH receptors during the prepubertal stage would therefore render young males infertile or at least delay acquisition of fertility. In the absence of any pharmacological antagonists yet available for this receptor, the only way to target them appears to be active (or passive) immunisation. In a previous study (Remy et al., 1993), prepubertal male Balb/c mice were immunized against the recombinant N-terminal domain of the porcine LHR (pLHRECD) overproduced through the Baculovirus-insect cells system and effects on fertility assessed. Up to 75% reduction of male fertility in terms of progeny size, and 60% of sterile couplings were obtained when treated males were mated with untreated fertile females. Related to these effects, testosterone concentrations were reduced as anti-receptor antibodies increased. In addition, vaccination against pLHR at the prepubertal stage significantly delayed initiation of fertility in nonsterilized animals. In order to better understand the mechanisms involved, a detailed histological study of treated versus untreated mice testis tissues was undertaken; it clearly indicated that all parameters reflecting spermatogenetic activity (Hochereau de Reviers, et al., unpublished observations) were affected. In this paper, data obtained from prepubertal rats of both sexes vaccinated against pLHR-ECD, and data from prepubertal male mice immunized against a pLHR peptide implicated in hormone recognition are discussed.

2. Methods

2.2. Immunization and mating protocols For immunization of Wistar rats against pLHRECD, the protocol described previously for Jico Balbl c mice (Remy et al., 1993) was used, except that males and females were treated. Briefly, 3 weeks before puberty, a primary immunization with insect cells (2 X IO6per animal) overproducing the pLHR-ECD in complete Freund adjuvant (CFA) was performed, and followed by one booster with 10 pg of purified antigen in incomplete Freund adjuvant (IFA) 3 weeks later. One week after booster, animals, now at the age of puberty, were mated during a week with untreated partners. Controls were injected in the same way with nonproducing insect cells and buffer in adjuvant. Fertility was assessed by counting the number of progeny. After weaning of the first set of progeny, that is about 2 months after the booster, the same pairs were allowed to mate a second time. Animals of both sexes from the first set of progeny born from immunized females (and untreated males) or untreated females (and immunized males) were allowed to mate with untreated partners in order to assess their fertility. For immunization of 3-week-old male Balb/c mice against the pLHR oligopeptide coupled to KLH, three intra-peritoneal injections were performed, at 2-week intervals, with 20 pg antigen in CFA per mouse the first time, 20 pg antigen in IFA the second, and 10 pg in IFA the third time. In this case, control animals received, following an identical schedule, the same quantities of carrier KLH protein in adjuvant. Ocular blood was collected at the time of the first mating (a few days after booster) and antibody levels in sera against the pLHR-ECD or the oligopeptide were evaluated by specific ELISA against the respective antigens.

2. I. Antigens

3. Results and discussion Recombinant extracellular domain of the porcine LH receptor, pLHR-ECD (amino acids l-370) was overproduced in insect cells and purified as described (Remy et al., 1993). The pLHR oligopeptide 20-36 was synthesized using a solid phase method, HPLC purified, and cystein coupled to keyhole limpet hemocyanin (IUH) .

3.1. Active and ‘passive’ immunisation ofprepubertal

rats Results are summarized in Fig. 1 where means ( f SE) of progeny per animal ( 12 matings per group) are shown. At the first mating (Fig. 1, first mates), a week after the booster, male fertility was affected as

J.-J. Rerny et al. /Livestock Production Science 42 (1995) 207-211

First Males

mate Females

Fl

from Males

immunized

animals

Females

IO I -

1 T

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animals). Offspring of both sexes were therefore raised without any treatment until puberty, and then mated. Interestingly, the fertility of male or female rats born from anti-pLHR immunized females was affected as if they had been actively immunized against pLHR themselves (5.3 f 1.5 for males, and 7 + 1.6 for females). As expected, animals born from untreated females and vaccinated males were not affected (8.4 + 1.2 for both sexes). 3.2. Anti LH receptor vaccination as an immunocastrative strategy: rational design of LH receptor antagonists

Fig. 1. First mate: the number of progeny per mate (mean f SE, tz= 12 for each group) obtained between rat males or females actively immunized against the recombinant N-terminal domain of porcine LH receptor (pLHR-ECD) or vehicle (control) and untreated partners is shown. Fl from immunized animals: the number of progeny per mate (mean f SE, n = 12 for each group) obtained between rat males or females born from couples in which males or females were immunized against pLHR-ECD.

the control group (7.6 + 0.9) gave birth to 30% more pups than the male vaccinated group (5.4 + 1.3). Female fertility was also affected, but to a lower extent (7 + 2 as compared to 6.1& 1.4). When the same animals were remated without antigen challenge between the two matings, no differences were observed between control and vaccinated animals (data not shown). Since females were also vaccinated against the LH receptor, we were able to compare fertility of the progeny born from treated females (mated with untreated males) and those from untreated females (mated with treated males), in order to see whether maternal antiLH receptor antibodies could have had any effect on the fertility of progeny (Fig. 1, Fl from immunized

Although development of male and female immunocontraceptive methods is of importance for mankind, there is an obvious need for low cost immunocastration strategies in livestock. Mechanical castration of young piglets is commonly done in normal breeding in order to prevent appearance of undesirable odorant substances, such as androstenone or skatole (Bonneau, 1982). This painful procedure is likely stressful, and results in growth deficit. Most of the approaches for lowering fat androstenone levels in male pig involve the use of agonists or antagonists for gonadotrophin or gonadotrophin-releasing hormones (Falvo et al., 1986; Dufour et al., 1993; Oonk et al., 1993) or immunization against them. In pig, the expression of LH receptor can be detected very early in fetal gonads of both sexes (Goxe at al., 1993)) persisting throughout life in males while disappearing rapidly in females until sexual maturity occurs. The results we obtained in rodents make the anti-LH receptor strategy potentially usable as a new alternative to mechanical castration: antagonizing LH receptors during the prepubertal stage could sufficiently inhibit and/or delay testicular maturation (therefore androstenone production) to suppress the need for castration. Since the recombinant LH receptor we overproduced was of porcine origin, it may be recognized as a self antigen by the porcine immune system, and may not induce comparable inhibiting activity as in rodents. In order to rationally design more efficient and specific LHR antagonist tools, information on important regions for hormone-receptor interactions and receptor activation are needed. For this purpose, different synthetic pLHR oligopeptides and antibodies raised against them were tested in vitro for their hormone inhibiting (binding and transduction) properties

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Table 1 Fertility, delayed births and sterility in Balb/c male mice immunized against pLHR peptide 20-36

Progeny from 7 pairs Mean * SE Fecundity Births: On time Late No birth

Controls

Immunized

42 6f0.57 6

21 3* 1.1 (P=O.O2) 5.25

417 317 o/7

217 21’7 317

3-week-old male mice were immunized as described in Methods with the peptide coupled to KLH (immunized) or KLH alone (controls). P value was obtained using the f test from Statview. Fecundity result from the total progeny divided by the number of pairs for each group. ‘On time’ and ‘late’ refers to births before or after the 22nd day following mating.

(Couture et al., 1994, submitted). Since oligopeptide 20-36 and anti-20-36 polyclonal antibodies were characterized as hormone binding inhibitors, the peptide was coupled to IUH, and used to immunise Balblc prepubertal male mice. Despite coupling to a carrier protein and two boosts, the KLH-coupled peptide was poorly immunogenic as compared to the whole receptor binding domain (data not shown). Nevertheless, as shown in Table 1, we found that the fertility of the peptide-KLH immunized male mice group was significatively affected. Moreover, as for mice vaccinated against pLHR-ECD, impaired fertility, as shown in Table 1, was mainly due to sterilization (3 17 sterile pairs at the first mating after immunization) and delayed initiation of fertility (2/7 on time deliveries in the peptide vaccinated group) rather than to a loss of fecundity. This result showed that it was possible to qualitatively reproduce (if not quantitatively, due to the poor antigenicity of the peptide) with a single peptide of 16 residues, the antifertility effects obtained with the whole pLHR binding domain.

4. Conclusions - Immunization of prepubertal male mice against the LH receptor or against a peptide involved in hormone binding to the LH receptor seems to mostly delay acquisition of fertility.

- In the rat, as opposed to the other mammalians species, FSH is essential for the initiation of spermatogenesis, which could explain why male rats fertility was less affected when vaccinated against LHR at the prepubertal stage. - Maternal anti-LHR antibodies seemed to have been able to produce the same antifertility effects than an active immunization at the prepubertal stage. Therefore, antagonizing gonadotrophins activities by the mean of immunization against functionnaly characterized domains of their receptors would be a way to immunomudulate steroidogenesis and fertility in different species.

Acknowledgements We thank Mr Cahier, Pellan, and Vigneau for their help in rats maintenance, and Mr Cayron for the care of mice. Part of this work was financed by the CRITTISIS, Nouzilly, France.

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