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This Article Abstract Full Text (PDF) All Versions of this Article: 68/1/323    most recent biolreprod.102.003699v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Abdennebi, L. Articles by Remy, J. J. Search for Related Content PubMed PubMed Citation Articles by Abdennebi, L. Articles by Remy, J. J. Agricola Articles by Abdennebi, L. Articles by Remy, J. J.

Maintenance of Sexual Immaturity in Male Mice and Bucks by Immunization Against N-Terminal Peptides of the Follicle-Stimulating Hormone Receptor¹

^a Laboratory of Molecular and Cellular Biology, I.N.R.A. Biotechnology, 78352 Jouy-en-Josas, France ^b Laboratory of Molecular and Cellular Biology, Inner Mongolia Agriculture University, 010018 Huhhot, China

	ABSTRACT

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The follicle-stimulating hormone is one of the two pituitary hormones that control fertility in both sexes. In the male, receptors for FSH (FSHR) are only expressed on testicular Sertoli cells. FSH plays different roles during the male life; it functions as a growth factor during development and sustains spermatogenesis in adults. However, the exact role of this hormone as an initiator of male fertility is not fully understood and few data are available concerning its involvement during the peripubertal period. We recently produced filamentous phages displaying FSHR fragments overlapping residues 18–38, which, if injected in animals, induced anti-FSH receptor immunity capable of inhibiting hormone binding. We employed this strategy to transiently inhibit FSH activity in male mice and male goats of the Saanen and the Mongolian Alpas Cashmere breeds at the prepubertal stage. Anti-FSHR peptide immunization from the age of 3 wk delayed the acquisition of fecundity in male mice by up to 1 wk. Once fertile, progeny sizes produced by mating immunized males and untreated females were found to be reduced by up to 60%. In two different breeds of goats, FSHR peptide vaccines were able to maintain circulating testosterone at low prepubertal levels for several months despite no alteration in LH levels, reflecting their ability to delay the onset of puberty. These results support the conclusion that FSH may play a central role in the male at puberty through the control of testosterone production.

follicle-stimulating hormone receptor, male sexual function, puberty

	INTRODUCTION

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In mammals, FSH has been considered as the main support of gametogenesis in both sexes, while LH is thought to control sex steroid biosynthesis. However, the respective roles of these two pituitary glycoprotein hormones in promoting fertility during development and puberty and in maintaining it at the adult stage have not yet been definitely delineated. Both gonadotropic hormones play essential roles during the development of male gonadal structures yet with probably significant differences between mammalian species. In men, an absence of LH activity (LH resistance) during development due to inactivating mutations in the LH receptor gene induces Leydig cell hypoplasia and sterility. Conversely, dramatic precocious puberty has been described in the case of LH receptor constitutive activation due to a gain in function mutations [1, 2]. An absence of FSH during development may lead to hypogonadism and azoospermia [3], and a few cases of hypergonadotropic hypogonadism have been linked to FSHR gene inactivating mutations in men [4, 5]. A number of experimental data have provided evidence that FSH may be an absolute requirement for sustaining spermatogenesis in human and nonhuman primates [6, 7]. However, in male mice, the observed phenotypes resulting from genetic inactivation of the beta subunit of FSH [8] or its receptor [9] appear to be slightly different; male mice without the functional hormone were reported to be fertile at the adult stage, while male mice with an inactivated receptor gene were affected in terms of their testicular differentiation and growth. They later exhibited a significant decrease in testosterone production, delayed puberty, and impaired spermatogenesis. Nevertheless, their fertility was only diminished at the adult stage [10–12]. Apart from their role in sustaining the normal growth of gonadal tissues during development, gonadotropic hormones are also important at puberty [13]. The acquisition of male fertility at puberty clearly involves considerable androgen synthesis concomitant with the initiation of spermatogenesis [14]. The endocrinology of puberty is not fully understood, and almost nothing is known about the cross-talk between the two gonadotropic hormones during this critical peripubertal period. This is essentially due to the lack of experimental systems to allow the efficient and specific transient impairment of the activity of one or other of the hormones. Recently, we constructed recombinant filamentous phages displaying overlapping peptides of the FSH receptor and used them as peptide vaccines [15]. Antibodies against these recombinant phages bearing FSH receptor sequences were shown to inhibit hormone binding and receptor activation in vitro, and their potential use as FSH antagonists was demonstrated in vivo when injected in the ewe [16]. The aim of the present study was to use the same immunological method to interact with the FSH function at the receptor level in prepubertal males. We show here that an inhibition of FSH activity in male mice during the prepubertal period affected the onset of sexual maturation and mean fertility, suggesting that anti-FSHR peptide vaccination was able to mimic gene inactivation. In immature bucks from two different goat breeds, Saanen and Mongolian Alpas Cashmere (MAC), anti-FSHR peptide vaccination was able transiently to maintain low prepubertal levels of circulating testosterone without any effects on growth rates.

	MATERIALS AND METHODS

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Recombinant Phages Displaying FSH Receptor Peptides

The construction of FSH receptor fragment-bearing phages has been previously described [16]. Briefly, oligonucleotides with compatible PstI and HindIII ends were purchased from GENSET (Paris, France). They encoded three different overlapping decapeptides of the human FSHR N-terminal region, respectively, peptide SKVTEIPSDL (SKV, residues 18–27), peptide SDLPRNAIEL (SDL, residues 25–34), and peptide RNAIELRFVL (RNA, residues 27–38). Annealed oligonucleotides were ligated to the double-stranded replicative-form (RF) DNA of PstI-HindIII restricted phage f88-4. The transformation of female electrocompetent MC1061 Escherichia coli yielded tetracycline-resistant colonies. Recombinant p8 protein primary structures were deduced from sequencing the resulting recombinant phages by automated DNA sequencing (Applied Biosystems, Roissy, France) using a specific p8 primer. Nonrecombinant phage particles expressing the wild-type coat proteins (W) were used as controls in mice, while control bucks were only injected with the vehicle.

Overnight culture of a single colony of MC1061 E. coli transformed by recombinant or wild-type phage RF-DNA in 1 L of NZY medium (1% enzymatic casein hydrolysate, 0.1 M NaCl, 0.5% bacto-yeast extract, pH 7) containing 40 µg/ml tetracycline made it possible to recover approximately 100 mg of polyethylene glycol (PEG 6000)-precipitated phage particles from the supernatants. The solubilization of particles in phosphate saline buffer (50 mM NaH₂PO₄, pH 7.4, 150 mM NaCl) was at a concentration of 10 mg/ml [17]. Before they were used as vaccines, the phage solutions were sterilized by heating at 70°C for 15 min and UV inactivated.

Animals and Immunization Procedures

Three-wk-old immature, male Balb/c mice (Iffa Credo, l'Arbresle, France) were injected i.p. with 0.1 mg of the respective phages bearing different FSH receptor peptides or an equimolar mixture of the three, diluted in 0.1 ml sterile PBS without adjuvant (n = 6 for each group). Two booster injections were performed at 6 and 7 wk. After the final antigen challenge, the immunized males were housed with untreated adult females. The presence of an estrus was determined by vaginal examination. First, pairs (one per cage and six pairs for each experimental group) were left together for 2 wk, equivalent to four estrous cycles. For each pair, the day of delivery was recorded. Then, in order to evaluate the longer term effects on fecundity, the same pairs were left together for a period of 2 mo. The progeny from each pair was isolated and counted.

With young bucks, two successive experiments with two different breeds were performed. As shown in Figure 1, the experimental procedures had been approved by the French and Chinese Ministries of Agriculture as well as by the Ethical Committee for INRA. During the first experiment, Saanen bucks were kept on an experimental farm (INRA, Brouessy, France) and fed according to the usual recommendations. The bucks were injected with a sterile solution without adjuvant but containing 1 mg of recombinant phages (only the mixture of the three recombinant phages was injected in bucks; n = 4) or 1 mg of wild-type phages (control group; n = 4). Primary injections were administered at the age of 4 wk, and subsequent booster injections were given as indicated in Figure 1. Plasma samples were collected from the animals weekly, and they were also weighed once a week.

View larger version (23K): [in this window] [in a new window]   FIG. 1. Immunization protocols. In Saanen bucks, primary immunizations were performed at the age of 7 wk (April). Three antigen challenges were performed, as indicated by arrows, in May, June, and September. The experimental period of 30 wk, due to the absence of antigen challenge between June and September, were divided into three periods. The MAC bucks were injected from the age of 1 mo and received monthly booster immunizations during an experimental period of 40 wk

A second experiment involved 20 animals of the Mongolia Alpas Cashmere (MAC) breed: 10 were FSH receptor peptide immunized (with the three recombinant phage mixture) and 10 were injected with the vehicle (control group). Four-week-old MAC bucks were injected with the vaccine preparations emulsified in complete Freund adjuvant following a protocol for booster injections (as indicated in Fig. 1), which differed slightly from that used in Saanen bucks. Plasma samples were collected and the animals weighed before each injection. The length of cashmere hair was measured every month. All experiments on MAC bucks were carried out in the experimental farm at Etogeqi (Inner Mongolia Agriculture University, Peoples' Republic of China). The animals were maintained in pasture without food supplementation.

Immune Responses

The immune responses of male mice and bucks were determined at different times in sera using enzyme-linked immunosorbent assay (ELISA). Plates (96 wells) were coated with solubilized phages in 100 µl coating buffer (50 mM NaCO₃, pH 8.8) overnight at 4°C and blocked with 1% BSA in PBS (pH 7.4) to avoid nonspecific binding. Control or immune sera from experimental animals were diluted at 1/1000 in PBS with 1% BSA and added to each well in duplicate. After the wells had been thoroughly washed, they were incubated with biotinylated rabbit anti-sheep IgG complexed to streptavidine peroxidase used at a dilution of 1/1000 (Amersham, les Ulis, France) for 1 h at 37°C. The TMB kit (Kirkegaard and Perry Laboratories, Gaithersburg, MD) was used as the peroxidase substrate. Optical densities at 450 nm were then determined.

Determination of Testosterone, LH, and FSH Levels

Testosterone concentrations in unextracted sera were assayed in duplicate using a single antibody radioimmunoassay kit (CIS-BIO International, Gif-sur-Yvette, France). This testosterone radioimmunoassay displays a sensitivity of 0.1 ng/ml, a cross-reactivity of 7.8% with 5-dihydrotestosterone, and a negligible cross-reactivity with other steroids. The intra- and interassay coefficients of variation were lower than 5% and 10.2%, respectively. LH was assayed in duplicate using a heterologous, double antibody radioimmunoassay with a rabbit anti-ovine LH antiserum and caprine LH as standard. FSH was assayed in duplicate using a rabbit anti-ovine FSH antiserum and ovine FSH (reagents were kindly provided by Dr. A.F. Parlow [18, 19]).

Data Analysis

Data were analyzed using the SYSTAT program (version 5) on a PC. For mice data, analysis of variance was used to assess the effect of the vaccine treatment. Multiple comparisons were then performed for data with a significant (P < 0.05) main effect using Duncan multiple range procedures to compare individual means. The values are expressed as mean ±SEM or as mean and range. P < 0.05 was considered significant.

	RESULTS

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The immune responses of mice to these antigens have already been analyzed and correlations between antiphage and anti-FSHR peptide antibodies established [16]. Buck immunity was therefore monitored using antiphage ELISA. For Saanen bucks, as shown in Figure 1, antigen challenges were discontinued after the first 3 mo of immunization (period I) for a period of 2 mo (period II), and the animals were then reimmunized (period III). Periods I and III corresponded to higher titers than period II (0.43 ± 0.019, n = 32; 0.48 ± 0.03, n = 12; and 0.34 ± 0.03, n = 12, respectively). MAC animals were challenged at the indicated times without any interruption in order to maintain constant anti-FSHR peptide antibody titers from May to January. No significant differences were observed in antibody titers when the two breeds were compared (data not shown).

FSHR Vaccine Effects in Male Mice

Prepubertal male mice were immunized with the different recombinant phages and their fertility assessed by mating with untreated females for 2 wk. Two effects on male fertility could be observed.

The first effect of anti-FSHR vaccination was to increase significantly the delay between mating and birth of the first litter, as shown in Figure 2A. Animals were housed together for a period corresponding to four estrous cycles. A mouse estrous cycle being 4 days, deliveries for control pairs occurred between Day 19 and Day 23 after mating, depending on whether pregnancy occurred on Day 1 or Day 4 of the cycle, and was related to the full sexual maturity of males (21 days was used as the standard period). In contrast, when males had been vaccinated with FSHR peptide recombinant phages, the females with which they were mated gave birth with highly significant delays of 3–9 days (P < 0.05 for SKV; P < 0.01 for SDL, RNA, and the mix) compared with controls, depending on the type of vaccines used. However, there were no significant differences between vaccines with different FSHR peptide phages in terms of delaying male mouse fertility.

View larger version (47K): [in this window] [in a new window]   FIG. 2. A) Delay in fertilizing ability of male mice. Three-wk-old Balb/C male mice were primo-injected with the different recombinant FSHR peptide phages (SKV, SDL, or RNA) or a mixture of the three (SKV + SDL + RNA) or with nonrecombinant phages (control). After two booster injections at 6 and 7 wk, they were mated with untreated females during 2 wk (six males for each group). For control pairs, the time taken to deliver from mating was 21 ± 2 days. The results for FSHR-vaccinated groups were normalized to control groups and the means were statistically significant, with P < 0.05 for the SKV vaccine (*) and P < 0.01 for the other vaccines (**). B) Effect of FSHR peptide vaccines on litter size. Mean litter size from pairs including recombinant FSHR peptide phage or wild-type phage-immunized males (six males for each group). The mean number of pups per pair is indicated for the different groups. Only the vaccinated groups with recombinant RNA phages or with the mixture of the three recombinant phages gave significant differences in mean of litter sizes when compared with the control group (P < 0.05)

The second effect of anti-FSHR peptide immunization was on litter sizes, as shown in Figure 2B. Control pairs (W) gave birth to a mean of 6.1 ± 1.2 pups per pair, while all groups of FSHR peptide-immunized males displayed significant impairment of fecundity, giving birth to mean litter sizes of between 4.5 ± 0.5 and 2 ± 1, depending on the vaccine used. Statistically significant differences were observed (P < 0.01) with the RNA peptide vaccine and the vaccine made of a mixture of all three receptor peptides (P < 0.05) compared with wild-type phage-immunized animals. Previously, we had found no difference in testosterone levels between SKV-vaccinated and control groups [15].

Endocrine Effects in Bucks Vaccinated Against the FSH Receptor

It was clearly not possible to assess the effects of anti-FSH receptor vaccination on buck fertility through mating, as was the case for mice. Therefore, in bucks, the effects of FSHR immunization on the onset of puberty were measured by following the evolution of circulating testosterone levels throughout the experimental procedure. Testosterone concentrations were determined in both control and FSHR-vaccinated animals (Fig. 3). Testosterone levels were determined weekly in Saanen bucks for 28 wk or monthly in MAC bucks for 48 wk. After 1 mo of life, MAC bucks already displayed higher plasma testosterone levels than Saanen bucks, at 5.03 ± 2.55 ng/ml (n = 20) as compared with 0.78 ± 0.4 ng/ml (n = 8), respectively. Unexpectedly, testosterone time courses were found to be very different in the two breeds: in untreated Saanen bucks (Fig. 3A, control), despite a short peak during Weeks 5, 6, and 7, testosterone levels rose markedly from only the 18th week, reaching a maximum of 17 ng/ml at the age of 20 wk. They then declined and remained at less than 5 ng/ml from Week 23 to Week 28. In contrast, testosterone levels in unvaccinated MAC bucks (Fig. 3B, control) increased almost linearly from Week 4, starting from a value of about 5 ng/ml and reaching about 40 ng/ml at Week 32. They then fell to reach prepubertal levels over a period of approximately 10 wk.

View larger version (24K): [in this window] [in a new window]   FIG. 3. Testosterone levels in FSHR peptide-vaccinated bucks. Circulating testosterone levels during the anti-FSHR peptide immunization protocol for Saanen (A) and MAC (B) bucks were assayed by a specific RIA. Assays were performed every week for Saanen bucks and every month for MAC bucks. The mean testosterone level was compared between FSH receptor peptide-vaccinated and control groups at each sample time. Mean differences were found statistically significant (P < 0.01, *) or highly significant (P < 0.001, **) when indicated

Vaccination against FSHR totally abolished the first peak of androgen observed in control Saanen bucks (Fig. 3A, FSHR). Despite the fact that no antigen challenges were performed during phase II, the marked summer increase between Weeks 18 and 23 in control animals did not occur in vaccinated animals, as testosterone levels remained below the prepubertal level of 5 ng/ml. This strongly suggests a long-lasting effect of immunization on androgen levels because antireceptor antibodies decreased almost to preimmune levels during period II (not shown). In MAC bucks, anti-FSHR antibodies also strongly inhibited testosterone production from Week 10 to Week 26, after which testosterone levels in vaccinated animals remained significantly lower than in controls (Fig. 3B, FSHR) until Week 32. Then a dramatic decrease in testosterone levels occurred in all animals associated with or due to seasonal regulations.

LH and FSH concentrations were determined in Saanen buck blood samples during the three phases of immunization. Vaccination against FSHR never modified the circulating level of LH at any time during the experiment when compared with controls (0.34 ± 0.06 ng/ml and 0.39 ± 0.02 ng/ml for FSHR-vaccinated and control groups, respectively). FSH levels were found to be very low (<0.2 ng/ml) in all animals, and no detectable difference could be found between control and immunized animals.

Growth and Wool Production after FSHR Vaccination

Importantly, the treatment did not affect growth rate or cashmere production in MAC bucks. Differences were only observed when the two breeds were compared: Saanen bucks exhibited linear growth (y = 1.275x + 4.32; r² = 0.99), reaching about 50% of their adult body weight (90 kg) by the age of 50 wk, while MAC bucks grew more rapidly during the first 20 wk, reaching at this stage about 60% of their final body weight (40 kg), and then growing more slowly until the adult stage. MAC is one of the better Chinese cashmere-producing goat breeds. Cashmere hair growth follows a seasonal pattern, with hair length increasing during winter from September to March. The total cashmere production was evaluated in May after combing without separating the undercoat and guard hair. There were no significant differences between control and vaccinated groups in terms of hair length and cashmere wool production, with 510 ± 30 g and 523 ± 45 g for FSHR-vaccinated control animals, respectively.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The hypothalamus is thought to play a central role in sexual maturation. Among the events that bring about fertility, the regulation of GnRH secretion by TGF alpha and the erbB family may play an essential role in the initiation of puberty [20, 21]. It has been shown that the use of GnRH antagonists can influence the onset of puberty [13], although the involvement of the respective gonadotropic hormones during the pubertal period has not been well documented. The aim of this study was therefore to assess the specific contribution of FSH to the onset of male puberty in two different mammalian species. In the absence of pharmacological FSH antagonists, we used an antireceptor vaccination strategy, immunizing young immature males (3-wk-old mice and 1-mo-old bucks) against a functionally characterized FSH receptor region.

The effects observed after antireceptor immunization in mice largely mimicked those observed after genetic inactivation of the FSH receptor gene (FORKO mice): fecundity, as measured by litter size, was impaired in FORKO mice by about 30% [11], while we observed here a significant reduction in fertility (up to 60%) using specific anti-FSHR vaccines. A delay of about 20 days to achieve sexual maturity was observed in FORKO mice [11], while antireceptor vaccines were able to delay male mouse fecundity for more than a week. Interestingly, testosterone levels were significantly reduced in adult homozygous FORKO mice [10]. In adult monkeys, immunization with a recombinant FSH receptor preparation affects spermatogenesis, reducing the fertility index [22]. Gene inactivation, despite its highly informative nature, is only routinely feasible in mice; moreover, the phenotypes observed in adult animals result from the developmental effects of gene deletions. As described in adult monkeys [22], we showed during this study that, when possible, a strategy of immunoinactivation could be as efficient, with the advantage of being applicable to species in which gene knock-out technology is not applicable.

In this case, because of the choice of epitopes unique to the FSH receptor borne by the phages against which we immunized animals, we believe this vaccination to be as specific as gene knock out in inactivating FSH signaling. As an example, during this study, we demonstrated that transient inactivation of the FSH receptor was possible under normal breeding conditions for cashmere bucks in Mongolian pastures. To our knowledge, these findings provided the first insight into endocrine parameters in peripubertal bucks. Blocking FSHR retained young buck testosterone at prepubertal levels, delaying their sexual maturation for a period of 5–6 mo but not modifying LH signaling. The delay in testosterone secretion observed in FSHR-vaccinated bucks had no consistent effect on the initiation of cashmere hair growth, suggesting that testosterone does not appear to be associated with the regulation of cashmere production. In view of the increasing commercial interest in cashmere production during the mid-1980s, Chinese farms initiated a 10-yr phenotype selection program. Selection of the best male cashmere producers cannot be made before they are 2 yr old. During these 2 yr, many problems linked to male behavior may be encountered. Maintaining a prepubertal stage in young bucks without affecting the cashmere production therefore appears to be very necessary to breeders in Inner Mongolia. Moreover, a vaccine method that could reversibly delay male sexual maturation could be of general interest to the development of a contraceptive vaccine.

The direct or indirect mechanisms through which FSH plays a role in androgen synthesis is not fully understood: in FORKO mice, its effects could be interpreted as developmental defects leading to the abnormal activity of LH-responding Leydig cells [12]. Our data on young developed animals suggest that FSH may contribute to Leydig cell activity until puberty, at least in the two species studied. Whether this is indeed the case and to what extent in adult mature animals requires further investigation. The specific inactivation of gonadotropic hormone signaling through the use of antireceptor vaccines may be feasible in any mammalian species of either sex and at any time during life and could represent a valuable strategy in response to a number of fundamental questions in reproductive endocrinology.

	ACKNOWLEDGMENTS

 
We would like to thank Prof. G. Smith for generously supplying the 88-4 phages. We would also like to thank S. Canepa, C. Gauthier, and D. Andre (PRMD, INRA-Nouzilly, France) for their assistance in the radioimmunoassays of LH and FSH. The reagents for FSH assays were kindly provided by Dr. Parlow (NIH, USA). Warm thanks to the Brouessy Centre in France and the Etogeqi Centre in Inner Mongolia for their assistance with animal handling and management.

	FOOTNOTES

 
¹ This work was partially funded by a grant from the Association Franco-Chinoise pour la Recherche Scientifique et Technique (PRA BT 98-05).

² Correspondence: Jean Jacques Remy, NMDA, UMR 6156, Institut de Biologie du Développement de Marseille, Parc Scientifique de Luminy, case 907, 13288 Marseille Cedex 09, France. FAX: 33 0491269748; remy{at}ibdm.univ-mrs.fr

Received: 16 January 2002.

First decision: 6 February 2002.

Accepted: 5 August 2002.

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