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Laboratoire Mécanismes d’Action des Gonadotropines, Unité Mixte de Recherche 6073, Institut National de la Recherche Agronomique/Centre National de la Recherche Scientifique/Université de Tours, Station de Physiologie de la Reproduction des Mammifères Domestiques, 37380 Nouzilly, France
Address all correspondence and requests for reprints to: Dr. M. C. Maurel, Laboratoire Mécanismes d’Action des Gonadotropines, Unité Mixte de Recherche 6073, Institut National de la Recherche Agronomique/Centre National de la Recherche/Université de Tours, Station de Physiologie de la Reproduction des Mammifères Domestiques, 37380 Nouzilly, France. E-mail: maurel{at}tours.inra.fr.
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and ß, whose noncovalent association is required for their biological activities. Gonadotropins belong to the glycoprotein hormone family and comprise hypophysial LH and FSH, and human (hCG) and equine (eCG) chorionic gonadotropins. Gonadotropins are currently used in human and veterinary medicine to mimic the endocrine mechanisms of reproductive cycles. In humans, FSH and hCG are used to conduct in vitro fertilization protocols and in anovulation treatment. In ruminants, particularly goats and ewes, eCG is commonly used in association with a progestogen to induce and synchronize estrus and ovulation. eCG is a dimeric glycoprotein secreted by trophoblastic cells in the mare between the 36th and 120th d of gestation (1, 2). Interestingly, dimeric eCG binds only to the equine LH receptor (3, 4), whereas it exhibits pronounced FSH activity in addition to its LH activity in species other than equine (5, 6). Each subunit is composed of a peptidic part connected to a glycan moiety (N- and O-chains). eCG is the most heavily glycosylated glycoprotein hormone, with a majority of biantenna glycans ending mainly in sialic acids that play an important role in the hormone half-life in vivo (7). Glycan chains are also involved in the stability of the heterodimeric hormone (8) and are necessary for the efficiency of signal transduction (9, 10). The dual activity of eCG, its long half-life, and its availability in large quantities make this unique gonadotropin a convenient exogenous hormone in treatments to induce and synchronize estrus and ovulation in small ruminants.
However, the repeated use of eCG treatments for the induction of ovulation is generally followed by a decrease in fertility from 60% to 40% (11, 12). This phenomenon has been explained by unwanted immunological responses (13). Indeed, the presence of anti-eCG antibodies (anti-eCG Abs) in the plasma of eCG-treated goats and ewes has been demonstrated (11, 12, 13, 14). A survey of 2500 females (ewes and goats) demonstrated that a 500-IU eCG injection induced strictly similar humoral immune response kinetics, with a maximum anti-eCG Ab concentration between 10–17 d after injection (13, 14). About 60% of treated females developed Abs to eCG, but anti-eCG Ab levels were highly variable among individuals. Previous reports showed a significant association between the females displaying high or low anti-eCG response phenotypes and particular microsatellite alleles located inside the major histocompatibility complex class II (13, 14). Anti-eCG Abs, already present by the time of eCG injection and resulting from the previous immune response against eCG 1 yr earlier, were defined as residual anti-eCG Abs. Females with a high residual anti-eCG Ab concentration exhibited a significantly lower fertility after AI, because of a delay in both the onset of estrus and the preovulatory LH surge (13, 14).
Surprisingly, some females displayed significant fertility after AI despite high residual anti-eCG Ab levels. Therefore, to address the mechanism of immune interference in eCG-induced reproductive function, we tested the impact of anti-eCG Abs on both LH and FSH bioactivities of the eCG. In the present study we show that these polyclonal anti-eCG Abs could modulate one or both bioactivities of eCG differently. These anti-eCG Abs recognized primarily the glycan moiety of eCG. Furthermore, our results demonstrate that the interference of anti-eCG Abs with FSH activity and to a lesser extent with LH activity has a physiological impact on the fertility of treated females.
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Materials and Methods |
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For the present study we selected 37 goats that exhibited the highest anti-eCG Ab concentration on d 0 from 3–22 µg/ml and on d 10 from 6–70 µg/ml. The number of previous treatments per goat varied from 2–5, depending on the age of the female. The plasma control corresponded to a pool of plasmas from non-eCG-treated goats.
eCG (standard eCG FL 652), eLH, eCG - and ß-subunits, and eCG NZY-01 isoforms (15) were provided by Dr. Y. Combarnous and F. Lecompte (Institut National de la Recherche Agronomique, Nouzilly, France). Totally deglycosylated eCG (GRB-VII-128A) was provided by Dr. Georges R. Bousfield (Wichita State University, Wichita, KS).
Anti-eCG Ab affinity purification
Abs from selected plasmas were affinity-purified on a HiTrap G protein column (Amersham Pharmacia Biotech, Uppsala, Sweden) displaying a high binding capacity for goat immunoglobulins G (IgGs) (16). As previously described, 5 ml of each plasma were briefly submitted to affinity purification (13). The purified Abs were then stored at -20 C.
Measurement of anti-eCG Ab concentrations by a quantitative ELISA
The anti-eCG Ab concentrations in plasmas or IgG-purified fractions were measured using a specific quantitative ELISA as previously described (13). The anti-eCG Ab concentration was expressed as micrograms per milliliter of plasma.
LH in vitro bioassay
The LH in vitro bioassay was based on the stimulation of testosterone production by isolated rat Leydig cells as previously described (17). Different concentrations of eCG (100 µl) were preincubated overnight at room temperature, either with or without undiluted plasma (9 µl) or the corresponding IgG fraction, added to 91 µl Leibowitz L15 medium. Leydig cells (15 x 104 cells in 200 µl L15) were pooled with the preincubated samples and incubated for 4 h at 34 C. After centrifugation, the supernatants were stored at -20 C. Testosterone concentrations were assayed by a specific RIA (18).
FSH in vitro bioassay
The FSH in vitro bioassay was based on the stimulation of progesterone production by the Y1 cell line derived from a mouse adrenal cortex tumor stably expressing the human FSH receptor (donated by Ares Serono, Geneva, Switzerland) as previously described (19). Different concentrations of eCG (500 µl) were preincubated overnight at room temperature, either with or without 45 µl undiluted plasma or the corresponding IgG fraction added to 455 µl culture medium. Next, 15 x 104 Y1 cells were stimulated with 400 µl preincubated samples for 4 h at 37 C. The media were harvested, boiled, and centrifuged, and the supernatants were collected and assayed for progesterone by a specific RIA (20).
Sensitivity of LH and FSH in vitro bioassays
The sensitivities of the two biological assays obtained with Y1 cells and Leydig cells were analyzed with eCG ranging from 6–50 ng/ml. The progesterone secretion varied between 6.93–33.49 ng/ml, and the testosterone secretion ranged from 9.34–25.67 ng/ml. Progesterone and testosterone secretion started with the same concentration of eCG, i.e. 6 ng/ml. The same range of eCG stimulation was thus used for both in vitro biological assays.
Measurement of anti-eCG plasmas and IgG effects on LH and FSH bioactivities of eCG
In both biological assays we used plasmas collected on d 0 or 10 and their corresponding IgG fractions. Two controls were systematically performed. The first determined the basal level of hormone production by Leydig cells and Y1 cells. The second determined the level of hormone production by Leydig cells and Y1 cells in the presence of control plasma or of the corresponding IgG fraction devoid of anti-eCG Abs.
The results were expressed as the percentage of eCG bioactivity: 100 x [(A - B)/(C - D)], where A is the amount of progesterone or testosterone secreted during stimulation with a defined concentration of eCG preincubated with a plasma or IgG of treated females, B is the amount of progesterone or testosterone secreted during stimulation with preincubated plasma or with IgG of treated females without eCG, C is the amount of progesterone or testosterone secreted during stimulation with a defined concentration of eCG preincubated with a control plasma or with control IgG of untreated females, and D is the amount of progesterone or testosterone secreted during stimulation only with control plasma or with control IgG of untreated females.
Radioligand receptor assay
Only IgG fractions purified from anti-eCG plasmas collected on d 0 were analyzed. The ability of anti-eCG IgG to modulate the binding activity of eCG to the rat LH and FSH receptors was analyzed using a radioligand receptor assay, performed as previously described (17). Enriched receptor membrane fractions were prepared from rat testes. For the binding assay, 5 ng/ml to 10 µg/ml eCG were preincubated overnight at room temperature with 6.5 µg/ml anti-eCG Abs. Each preincubated sample (100 µl) was then incubated for 4 h at 34 C, with 50 µl buffer containing 36 mM CaCl2, 50 µl radioiodinated hormone (40,000 cpm) and 100 µl testicular membranes. The reaction was stopped by adding 2 ml cold 10 mM Tris-HCl, pH 7.4, at 4 C, and the tubes were centrifuged at 3,000 x g for 40 min. The bound radioactivity was measured with a 
-counter (Packard Instruments, Perkin-Elmer, Les Ulis, France).
Affinity measurement of anti-eCG Abs by surface plasmon resonance (SPR)
The anti-eCG Ab/eCG interaction was measured by SPR using a Biacore 1000 system (Biacore International, Uppsala, Sweden). Affinity measurements were performed with diluted plasmas or corresponding affinity-purified IgGs collected on d 0 or 10. Sensor chips, amine coupling kit, and HEPES-buffered saline (HBS) buffer were supplied by Biacore (21).
eCG (35 µl) prepared at 100 µg/ml in 10 mM sodium acetate, pH 4, was covalently coupled on either a carboxymethylated dextran CM5 sensor chip for purified IgGs or an F1 sensor chip for plasma analysis, using an amine coupling kit as described by the manufacturer. F1 sensor chips are well suited for plasma samples because their dextran matrix is shorter than CM5, and this reduces nonspecific binding when diluted plasmas are injected. The eCG immobilization level was 6500 resonance units (RU) on CM5 and 1600 RU on F1 chips.
Kinetic measurements of anti-eCG Ab interaction on coupled eCG were performed with HBS as running (20 µl/min) and diluting buffers at 23 C. The anti-eCG Ab concentration of injected IgG fraction (35 µl) ranged from 1.2–64 nM. Injected plasmas (35 µl) were diluted from 1:10 to 1:80. To prevent nonspecific interactions on the coated surface, purified IgGs and plasmas were preincubated for 30 min at 37 C in HBS to which was added 1 mg/ml CM-dextran (Fluka, Buchs, Switzerland) before injection. The regeneration of the coupled surface was performed by injecting NaOH 10 mM (10 µl). The binding curve obtained with control plasma or purified IgG fraction was used for nonspecific binding subtraction.
Kinetic and affinity constants were calculated using BIA Evaluation software (version 2.2.4). This Biacore software allows kinetic constants (kon and koff) to be calculated with SD and a statistical validation test (
2). The affinity constant was calculated as the kon/koff ratio, and the dissociation constant was calculated as the koff/kon ratio.
Specificity of anti-eCG Abs
The eCG regions recognized by anti-eCG Abs were determined using a competitive ELISA, previously described (22). Before the assay, goat plasmas collected on d 0 or 10 were incubated overnight with increasing concentrations (from 50 ng/ml to 10 µg/ml) of standard eCG FL652, isolated eCG - and ß-subunits, eLH, totally deglycosylated eCG (dg eCG), eCG treated with neuraminidase (eCG SA4, 1% sialic acid), and three isoforms of eCG NZY-01 (15): eCG isoform with 17% sialic acid (eCG SA1), eCG isoform with 9.5% sialic acid (eCG SA2), and eCG isoform with 4.7% sialic acid (eCG SA3). The 100% cross-reaction was obtained with standard eCG FL652.
Statistical analysis
Average values were represented as the mean ± SEM. Differences in progesterone and testosterone secretions were determined by t test or ANOVA. Differences were considered significant at P 
0.05.
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shows the effects of plasmas on the LH and FSH bioactivities of eCG, ranging from 6–50 ng/ml, incubated with medium, with plasmas from untreated females, or with different plasmas from eCG-treated females. For each assay, eCG preincubated with medium or control plasma was used as a reference. Both controls led to identical testosterone and progesterone secretion when stimulation was carried out with a similar concentration of eCG. However, when eCG was preincubated with plasmas containing anti-eCG Abs, three different effects on LH and FSH bioactivities of eCG were observed. First, some plasmas displayed no effect on LH and FSH bioactivities and induced the same testosterone and progesterone production as that obtained with the control plasma. Secondly, some plasmas displayed an inhibitory effect on LH and/or FSH bioactivities. These plasmas induced lower progesterone and testosterone secretion for all eCG concentrations tested compared with the control plasma. In the last case, some plasmas displayed a hyperstimulatory effect on LH and/or FSH bioactivities, leading to higher testosterone and progesterone secretion for all eCG concentrations tested compared with control plasma. By contrast, when incubated without eCG, all of these plasmas had no significant effect on basal secretion of progesterone and testosterone.
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), 2 plasmas inhibited up to 10 times only FSH bioactivity (Fig. 2B), and 4 plasmas inhibited up to two times only LH bioactivity (Fig. 2C). Surprisingly, three other types included plasmas displaying a hyperstimulatory effect on the eCG bioactivities (n = 10): two plasmas hyperstimulated both LH and FSH bioactivities (up to 2.3 times for LH bioactivity and up to 1.5 times for FSH bioactivity; Fig. 2D), four plasmas hyperstimulated up to 2 times only FSH bioactivity (Fig. 2E), and four plasmas hyperstimulated up to two times only LH bioactivity (Fig. 2F). These results demonstrate that plasmas of eCG-treated females containing anti-eCG Abs can modulate in different ways one or both bioactivities of eCG.
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When the inhibitory plasma or its purified IgG fraction was preincubated with 100 ng/ml eCG, testosterone secretion decreased to 28% (Fig. 3A) and 31% (Fig. 3B) of the control level, respectively, and progesterone secretion to 20% (Fig. 3C) and 53% (Fig. 3D) of the control level, respectively. When the hyperstimulatory plasma or its purified IgG fraction was preincubated with 100 ng/ml eCG, testosterone secretion increased to 145% and 156% (Fig. 3, A and B) of the control level, respectively, and progesterone secretion to 144% (Fig. 3C) and 171% (Fig. 3D) of the control level, respectively. The non-IgG counterpart of the plasmas did not modify LH and FSH bioactivities (data not shown).
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Modulation of LH or FSH bioactivity of eCG by anti-eCG Abs affects the fertility of treated goats differently
It has been demonstrated that only residual anti-eCG Abs originating from the previous treatment impact on the decrease in fertility. Females with high residual anti-eCG concentration exhibit a lower fertility after AI because of a delay in the preovulatory LH surge. As demonstrated above, these residual Abs displayed an effect on the LH and/or FSH bioactivities of eCG.
For this study we analyzed anti-eCG plasmas (n = 37) collected on d 0, just before the administration of a new eCG treatment. All exhibited a high anti-eCG Ab concentration. Our aim was to discriminate which effect of anti-eCG plasmas on LH and FSH bioactivities had the greatest impact on the fertility of treated goats after AI. We compared the effects of the plasmas on 12.5 ng/ml eCG, which roughly corresponds to the quantity of injected eCG (50 µg), taking into account the blood volume of a goat (4 liters). Table 1 shows the effects of anti-eCG plasmas on the eCG bioactivities and fertility results of females after the newly administered eCG treatment and AI. Eleven goats were not pregnant, and 26 were pregnant and kidded. First, we observed that 100% of goats with anti-eCG Abs with a hyperstimulatory effect on FSH bioactivity were pregnant and kidded, as did 76% of goats with anti-eCG Abs with no effect on FSH bioactivity. By contrast, none of the goats with anti-eCG Abs inhibitory of FSH bioactivity was pregnant. However, among the females with anti-eCG Abs inhibitory of LH bioactivity, only 39% were pregnant and kidded. This result contrasts with 91% of pregnant females in which anti-eCG Abs had a hyperstimulatory effect on LH bioactivity and 85% of pregnant females in which anti-eCG Abs had no effect on LH bioactivity. For comparison, the normal pregnancy rate when goats are treated with eCG for the first time is, on the average, 65%. Our results demonstrate that plasmas displaying a hyperstimulatory effect on both bioactivities only come from pregnant goats, and plasmas displaying an inhibitory effect on both bioactivities only come from nonpregnant goats. This study explains the lack of pregnancy of treated females due to an inhibition of eCG bioactivities by anti-eCG Abs, especially Abs inhibitory of FSH bioactivity. These results clearly demonstrate that the FSH bioactivity of eCG and, to a lesser extent, LH bioactivity are necessary to induce ovulation with accurate timing to achieve significant fertility of treated and inseminated females.
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, A and B). The first inhibitory IgG fraction elicited 48% and 45% inhibition of binding to FSH and LH receptors, respectively. The second inhibitory IgG fraction did not alter the binding of eCG to either receptor, as the binding curve obtained was similar to that of eCG preincubated with medium alone. Similarly, we investigated the effect of an IgG fraction hyperstimulatory of both eCG bioactivities, which did not change the binding of eCG to the FSH and LH receptors.
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The results obtained showed that plasmas or IgG fractions displayed strikingly different interaction kinetics whatever their effects on the eCG bioactivities. For example, when samples were analyzed at 13.3 nM anti-eCG Ab, the maximal binding level, measured just after the injection of samples, varied from 150-1200 RU. Likewise, the rate of dissociation measured 6 min later varied from 10–70% (data not shown).
The affinity measurements of four types of anti-eCG Abs are summarized in Table 2. The results obtained clearly show a high variability in the affinity (Ka) and dissociation constant (Kd) values whatever the effects of anti-eCG Abs on eCG bioactivities. The dissociation constants varied from 0.06–13.3 nM. It should be noted that most of the tested plasmas or IgGs displayed a high affinity for eCG, with the Ka value ranging from 1010–108 M-1. Significantly, these results clearly demonstrated that anti-eCG Abs displaying a strong effect on eCG bioactivities did not have the highest affinity constant. Likewise, the lack of effect on eCG bioactivities was not due to a low affinity of anti-eCG Abs, but, rather, resulted from the localization of the epitopes they recognized on the hormone surface.
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-subunit, eCG ß-subunit, chemically dg eCG, or eLH. Cross-reaction was calculated at 50% of the displacement obtained with purified native eCG as competitor by competitive ELISA.
Table 3 summarizes the specificity of the plasmas in relation to their effects on eCG bioactivities. Most plasmas weakly recognized isolated eCG - and ß-subunits and dg eCG. Nineteen of 21 plasmas cross-reacted with eCG less than 5%, and the other 2 cross-reacted less than 9%. Eighteen of 21 plasmas cross-reacted with eCGß less than 1.5%, and the other 3 cross-reacted between 20–44.7%. Eighteen of 21 plasmas did not recognize dg eCG or recognized it only very weakly, with a cross-reaction of less than 5%, and the other 3 had a cross-reaction of less than 20%. Thus, plasmas containing anti-eCG Abs preferentially recognized the eCGß dimer and seemed to be influenced by the glycan chains of the dimeric hormone.
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and clearly cross-reacted with eCG ß-subunit (between 20–44.7%). Plasmas with an inhibitory effect on both eCG bioactivities weakly recognized dg eCG and eCG -subunit. No plasma displaying no effect on either eCG bioactivity recognized eCGß. For the other types of plasmas, we did not observe a correlation between the specificity and the modulation of eCG bioactivities by anti-eCG Abs. To confirm these observations, we analyzed the cross-reaction with eLH, which has the same peptidic structure as eCG, but differs in its glycan composition. We observed that anti-eCG Abs did not recognize eLH. These results confirm that most anti-eCG Abs recognize the glycan moiety of eCG.
The glycan part of eCG is mainly composed of biantenna glycans ending in sialic acids, in contrast with eLH ending mainly in sulfates. Therefore, we next tested plasma specificity toward totally dg eCG and different dg isoforms of eCG. We found that the sialic acid composition of eCG modulated the plasma specificity for eCG (Table 4), which decreased in line with the sialic acid content. Only 5 of the 10 plasma samples used in this study were tested against dg eCG (1% of sialic acids), and none of these five recognized the desialylated eCG. These data demonstrate the important role of sialic acids in the immunogenicity of eCG. They reinforce the importance of the specific glycan sequences of eCG, and especially sialic acids, in the humoral immune response induced by eCG treatment.
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Modulation of the biological activity of hormones by Abs, especially with monoclonal Abs (mAbs), has been reported in several in vitro or in vivo studies. In superovulation treatments, the administration of an anti-eCG mAb (Neutra-pregnant mare’s serum gonadotropin) shortly after the preovulatory LH surge synchronized final follicular maturation and shortened the period of multiple ovulations by neutralizing eCG (25). Other reports have shown an enhancement of bioactivity for human, bovine and porcine GH (26, 27, 28) and human TSH (29) by complexing with certain mAbs. In vivo, the potentiation of GH (26) and TSH (29) activity by mAbs has been demonstrated in hypopituitary Snell dwarf mice as well as in normal sheep for GH (30). Our results are reinforced by a previous study (31) with 14 anti-eCG mAbs showing that some anti-eCG mAbs modulated LH and/or FSH bioactivities of eCG either positively or negatively. The mAbs without an effect on eCG bioactivities had the lowest affinity for the hormone, and the degree of inhibition of eCG bioactivities was correlated to increasing mAb affinity for eCG. By contrast, our results showed that the level of modulation of eCG bioactivities did not correlate with the affinity of polyclonal anti-eCG Abs present in treated goats. Thus, the highest affinity for eCG was observed for anti-eCG Ab without an effect on eCG bioactivities.
In the present report we show that the modulating effect of anti-eCG Abs on eCG bioactivities can be explained by two mechanisms. Some anti-eCG Abs blocked hormone interaction with its specific receptors, probably because the anti-eCG Ab epitopes overlap the receptor-binding site on the eCG surface, inducing a steric hindrance that prohibits hormone binding. Other anti-eCG Abs did not disturb the eCG binding, probably because they could induce a conformational change in the three-dimensional structure of the hormone, leading to a specific enhancement or inhibition of its biological activity. Indeed, the enhancement of human TSH bioactivity can be explained by a modification of the hormone conformation by mAbs, which fits into the receptor better (29), whereas the enhancement of human GH bioactivity can be explained by an alteration of the nature or of the duration of the interaction between the mAb-hormone-receptor complex (32).
The different anti-eCG Ab effects on one or another eCG bioactivity suggest the existence of different molecular determinants responsible for each of the LH and FSH bioactivities. This view reinforces the hypothesis of negative specificity (33, 34), which assumes that there is a high affinity site carried on the -subunit of all glycoprotein hormones and inhibitory sites located on the ß-subunit in position 94–96 for LH bioactivity and in position 102–103 for FSH bioactivity, which prevents the binding of dimeric hormones to other receptors (35, 36). Therefore, it is possible that some polyclonal anti-eCG Abs could modify the folding of the ß-subunit sequences of eCG and thus modulate LH and/or FSH bioactivities.
Our results concerning anti-eCG Ab specificity demonstrated that anti-eCG Abs from treated goats weakly recognized eCG ß- and -subunits and did not display common specificity profiles according to their effects on eCG bioactivities. Surprisingly, our study showed that none recognized native eLH. However, eCG and eLH have identical polypeptidic sequences, encoded by the same - and ß-subunit genes (37, 38). The major structural differences are the glycosylation of the N- and O-glycan chains linked to the - and ß-subunits of eCG. Glycan chains on eCG are mainly terminated by sialic acids, whereas more than 72% of glycan chains on eLH end in sulfates and a few in sialic acids (39, 40). We demonstrated that sialic acids are very important in the immunogenicity of eCG, because the cross-reactivity of plasmas containing anti-eCG Abs decreased in line with the sialic acid content of eCG. The majority of animals possess two types of sialic acids, N-acetyl-neuraminic acid and N-glycolyl-neuraminic acid (Neu5Gc). However, Neu5Gc is absent only in adult birds and humans (41). Injections of glycoconjugates containing Neu5Gc are highly immunogenic in adult humans, and exposure of humans to horse serum results in a serum sickness reaction (42, 43). Two other types of sialic acids (4-O-acetyl-9-O-lactyl-N-acetyl-neuraminic acid and 4-O-acetyl-9-O-lactyl-N-glycolyl-neuraminic acid) have only been identified in horses (41). This difference in sialic acids could explain the immunogenic properties of eCG when injected in goats. Thus, anti-eCG polyclonal Abs largely seem to recognize glycan chains, but we cannot exclude that anti-eCG Abs recognize a peptidic moiety of the hormone only when modeled by the glycan environment. Indeed, an mAb specific for the hCGß 109–145 region recognized a delineated epitope strictly dependent on a glycan environment in region 115–145 (44). This epitope was recognized to an extent on native hCG, whereas it was not recognized on desialyated hCG.
Numerous studies have reported the importance of glycan chains in the hormonal signal transduction (45, 46, 47). For example, the presence of Asn52 glycan in the hCG dimer is necessary for inducing and stabilizing a conformational change in hCG upon binding to the receptor, resulting in activation of signal transduction pathways (48, 49). Thus, a direct interaction of Abs on one or several glycan chains could provoke a total inhibition of the bioactivity by steric hindrance. Another possibility would be that the binding of Abs to one of the glycan chains would freeze the latter in a given position and induce a stimulation of the hormonal signal by transconformation of the hormone. Such Abs would be very valuable tools for studying the interference mechanism of the hormone glycan chains in the transduction of the hormonal signal.
In the present study we show that residual polyclonal anti-eCG Abs could modulate one or both eCG bioactivities differently. Both LH and FSH bioactivities of eCG are important for the efficacy of the treatment, because FSH in mammals induces follicular growth and maturation, whereas LH leads to terminal follicular growth and ovulation. The real importance of FSH bioactivity in ovarian stimulation and fertility is supported by transgenic mice whose FSH ß-subunit or FSH receptor genes have been invalidated, presenting a blockade of folliculogenesis before the preantrum stage, resulting in female sterility (50, 51). The comparison of anti-eCG Ab effects in relation to the fertility of treated goats after AI proves that FSH bioactivity and, to a lesser extent, LH activity are indispensable for efficient ovarian stimulation and subsequent fertility.
The present results lead us to consider the use of anti-eCG Abs to modulate, either positively or negatively, the biological activity of exogenously injected hormones. These Abs could be valuable in diminishing the quantity of injected eCG while still obtaining significant fertility in ovulation induction treatments. Such Abs are particularly interesting and have implications for animal husbandry as well as potentially for human fertility treatment and assisted reproduction techniques, where exogenous gonadotropins are intensively used.
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Acknowledgments |
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-eCG and ß-eCG subunits, and eCG NZY-01 isoforms and for their critical comments and many helpful discussions. We are grateful to Dr. G. Bousfield (Wichita University, Wichita, KS) for providing totally deglycosylated eCG GRB-VII-128A. We thank A. Ythier (Ares Serono, Geneva, Switzerland) for the gift of Y1 cells stably expressing the human FSH receptor. We acknowledge F. Bouvier (Institut National de la Recherche Agronomique, Domaine de Galles, Avord, France) and all the breeders involved in this study. We also thank Dr. P. Crepieux for critical reading and for reviewing the English of the manuscript, and Dr. H. Watier for scientific comments. |
Footnotes |
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Present address for F.R.: International Agency for Research on Cancer, 150 Cours Albert Thomas, 69008 Lyon, France.
Abbreviations: Ab, Antibody; AI, artificial insemination; dg, deglycosylated; eCG, equine chorionic gonadotropin; HBS, HEPES-buffered saline; hCG, human chorionic gonadotropin; IgG, immunoglobulin G; mAb, monoclonal antibody; Neu5Gc, N-glycolyl-neuraminic acid; RU, resonance units; SA, sialic acid; SPR, surface plasmon resonance.
Received May 14, 2003.
Accepted for publication September 26, 2003.
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- and ß-subunits. Biochim Biophys Acta 1159:74–80[CrossRef][Medline]
2,3Gal, respectively. J Biol Chem 268:795–802 subunit in transduction of biological signal in glycoprotein hormones. Science 229:65–67-subunit of human chorionic gonadotropin in the ß dimer. Biochem J 364:485–495[CrossRef][Medline]
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), no effect on either LH or FSH bioactivities (
), or a hyperstimulating effect on both LH and FSH bioactivities (
) and with eCG preincubated with control plasma (
).
) of eCG. The dark line corresponding to 100% bioactivity was obtained with control plasma of untreated females, devoid of anti-eCG Abs (-). The different histograms represent a plasma displaying an inhibitory effect on both LH and FSH bioactivities (A), a plasma displaying an inhibitory effect only on FSH bioactivity (B), a plasma displaying an inhibitory effect only on LH bioactivity (C), a plasma displaying a hyperstimulating effect on both LH and FSH bioactivities (D), a plasma displaying a hyperstimulating effect only on FSH bioactivity (E), and a plasma displaying a hyperstimulating effect only on LH bioactivity (F). Testosterone and progesterone secretion were measured as described above. **, Significant difference in bioactivity percentage (P < 0.05).
