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Endocrinology Vol. 147, No. 9 4205-4212
Copyright © 2006 by The Endocrine Society

Biological Properties of a Novel Follicle-Stimulating Hormone/Human Chorionic Gonadotropin Chimeric Gonadotropin

Louise M. Garone, Elena Ammannati, Theresa S. Brush, David J. Fischer, Enrico Gillio Tos, Jiangping Luo, Kimberley L. Altobello, Cinzia Ciampolillo, Thomas M. Ihley, Emmi Kurosawa, Angela Tiebout and Sean McKenna

Serono Research Institute, Inc. (L.M.G., T.S.B., D.J.F., J.L., K.L.A., T.M.I., E.K., A.T., S.M.), Rockland, Massachusetts 02370; Istituto di Ricerche Biomediche "Antoine Marxer" (E.A., E.G.T., C.C.), 10010 Colleretto Giacosa, Torino, Italy

Address all correspondence and requests for reprints to: Louise M. Garone, Serono Research Institute, One Technology Place, Rockland Massachusetts 02370. E-mail: louise.garone{at}serono.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A chimeric recombinant human gonadotropin, termed C3, demonstrates both follitropic and lutropic bioactivities. The {alpha}-subunit construct for C3 is comprised of the recombinant wild-type human glycoprotein hormone {alpha}-subunit. The ß-subunit DNA construct for C3 encodes residues 1–145 from human chorionic gonadotropin (hCG)-ß with the exceptions that FSHß amino acid 88 (D) is substituted for hCGß amino acid 94 (R) and FSHß amino acids 95–108 (TVRGLGPSYCSFGE) are substituted for hCGß amino acids 101–114 (GGPKDHPLTCDDPR). C3 is a potent FSH and LH agonist able to bind and to signal through FSH and LH receptors in vitro. In in vivo bioassays optimized to quantify each type of activity, C3 was found to have lutropin and follitropin potencies at levels similar to those of recombinant human LH and recombinant human FSH, respectively. In immature rats, C3 was sufficient to support the maturation of normal ovarian follicles. Moreover, a significant portion of follicles matured by C3 ruptured in response to an ovulatory hCG stimulus and gave rise to morphologically normal oocytes. Furthermore, a low dose of C3 promoted weight gain in the rodent uterus, suggesting it also supported preparation for implantation without histological evidence of excessive luteinization of the ovary. In summary, the biological properties of C3 indicate that its chimeric nature has resulted in a fully functional, dual-acting human gonadotropin.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FSH (FOLLITROPIN) AND LH (LUTROPIN) are pituitary glycoprotein hormones that contribute to the endogenous regulation of gamete production in vertebrates through interaction with FSH receptors and LH receptors, respectively, expressed on somatic cells in the ovary (1, 2, 3). Human chorionic gonadotropin (hCG) is a pregnancy-associated, placenta-derived protein hormone that, like the structurally related LH, interacts with the LH receptor-mediating lutropin activity (4). The first biotherapeutic used to promote gametogenesis in infertile women was a mixture of gonadotropins isolated from the urine of postmenopausal women; although such mixtures differed with respect to their ratios of follitropin to lutropin potency by up to 100-fold, these were deemed clinically acceptable as long as the patient received a sufficient total dose of FSH (5). Follicular development cannot occur in the absence of either FSH (6) or LH (7), but the stage at which follicle development arrests differs between genetically engineered FSH and LH knockout mice. In the ovaries of FSH knockout female mice, follicles fail to enter the preantral stage of development, whereas in the LH knockout mice, ovarian follicles fail to complete the antral stage of development. With the availability of GnRH antagonists and recombinant gonadotropins, it is possible to ascertain the precise contributions of FSH and LH in subfertile women. Several studies support the adjunct use of LH with FSH in certain patient populations (8, 9). Humaidan et al. (10) observed improved implantation rates and reduction in FSH usage in patients 35 yr old or older when FSH was supplemented with LH.

The ability of related gonadotropins to distinguish between LH and FSH receptors has been an area of active study. Campbell et al. (11) demonstrated that this ability in hCG resided in a portion of the ß-subunit that was carboxyl-terminal to the 10th cysteine (Cys) residue. Subsequently it was found that replacing a portion of the hCG ß-subunit amino acid sequence between the 11th and 12th Cys residues (see Fig. 1Go) with their FSH counterparts enabled the resulting chimera to bind both LH (LHRs) and FSH receptors (FSHRs) (12). hCG/FSH chimeras that contained FSH residues between ß-subunit Cys10 and Cys12 or Cys11 and Cys12 bound FSH receptors with roughly the same affinity, but the latter had much better ability to bind LH receptors. This indicated that differences in ß-subunits between Cys10 and Cys11, i.e. residues 94–97, might have greater influence on LH activity than FSH activity. This was confirmed by studies showing the ratio of LHR to FSHR agonist activities of these analogs in vitro could be varied by approximately 100-fold, simply by altering the composition and charge of residues 94–97 (12, 13). A few residues on the carboxyl-terminal side of cysteine 12 (hCGß 111–114) were also found to have a small influence on the FSH to LH activity ratio in vitro (14). The impact of these alterations on the relative follitropin and lutropin activities in vivo has not been evaluated. Based on these earlier studies, we generated a chimeric gonadotropin, termed C3, comprised of a wild-type {alpha}-chain and an hCG ß-chain, which contains aspects of some of these molecules, including substitution with corresponding FSH sequences between Cys 11 and 12 (hCG amino acid sequences 101–109) and sequences C terminal to Cys 12 (amino acids 111–114) as well as the first amino acid C terminal to Cys 10 (amino acid 94). We describe the physical and biological properties of this molecule in vitro and in vivo and discuss the possible clinical utility of such a dual-acting gonadotropin.


Figure 1
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  		FIG. 1. Amino acid sequence alignment of ß-subunits for hCG, hFSH, and C3. Conserved cysteine residues are bold and, in C3, amino acid sequence indigenous to hFSH is highlighted in gray, whereas sequence indigenous to hCG is not highlighted.

 

   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
C3 expression and purification
A construct encoding part of the C3 ß-subunit (pMB144) was generously provided by Dr. William Moyle (Department of Obstetrics and Gynecology, Robert Wood Johnson Medical School, Piscataway, NJ) and further modified to give the desired set of amino acid sequence changes, using PCR methods. The final product coded for a polypeptide homologous to the hCG ß-chain except for the substitution of amino acid D (aspartic acid) for R (arginine) at position 94 and amino acids TVRGLGPSYCSFGE substituted for hCGß residues 101–114 (GGPKDHPLTCDDPR). The C3 ß-subunit cDNA was then subcloned into XhoI site within pCI-neo vector (Promega, Madison, WI). The protein-coding region was verified by sequencing analysis (ABI Prism 310 genetic analyzer; Applied Biosystems, Foster City, CA).

The D{alpha} vector, used for stable CHO cell expression of the gonadotropin {alpha}-subunit, is a derivative of pCLH3AXSV2DHFR. The vector was modified to include a heterologous intron with a synthetic splice donor engineered upstream of the 2-kb XbaI-PstI fragment from intron A of the gonadotropin {alpha}-subunit gene (containing a splice acceptor). The heterologous intron is situated between the promoter and the XhoI cloning site, placing it in the 5' untranslated region of the RNA transcript. The D{alpha} vector has been described in more detail elsewhere (15).

A single T25 flask of CHO-DUKX cells was cotransfected with human {alpha}-subunit cDNA in D{alpha} and the C3 ß-subunit expression construct (1:1 ratio) in a small-scale lipofectamine 2000 protocol. Forty-eight hours after transfection, cells were split 1:40 into 10 100-mm dishes and fed with growth medium [MEM{alpha} (+), 10% fetal bovine serum, 4 mM L-glutamine]. The following day the pools were fed selection medium [MEM{alpha} (–), 10% dialyzed fetal bovine serum, 4 mM L-glutamine] containing 0.02 µM methotrexate (MTX) to begin the selection process. Gene amplification was induced with MTX by gradually increasing concentration (maximum 0.10 µM) as the pools were fed. After amplification, the expression levels were measured by Active hCG ELISA (10-8300; Diagnostic Systems Laboratories Inc., Webster, TX). Cells from the highest-expressing pool were cryopreserved. Cells from the initial bank were used for bioproduction and clone selection. All protein described in this paper was produced by that pool in cell factories out of MTX selection in serum-free medium. Six liters of conditioned medium were harvested containing, on average, 1.8 µg/ml.

C3 was purified by immunoaffinity chromatography using an anti-hCG monoclonal antibody (hCG09) ascites fluid generated at The University of Cambridge (Cambridge, UK). The immunoaffinity adsorbent consisted of monoclonal antibody hCG09, which had been purified by Protein A Fast Flow chromatography, coupled to Poros EP beads. The equilibration buffer was 0.1 M Tris (pH 7.4) containing 0.5 M NaCl. Twenty- to forty-fold concentrated culture supernatant was dialyzed exhaustively against this buffer before exposure to the column. C3 was eluted in a manner similar to that described for hCG (16), with 2 M potassium thiocyanate in 50 mM sodium phosphate buffer (pH 5.0). Immunoaffinity eluate fractions (2 ml) were collected into tubes containing 8 ml of 50 mM sodium phosphate buffer (pH 7.9) and further diluted with water to reduce the thiocyanate concentration and raise pH before concentration and analysis. This purification was conducted twice at Serono Research Institute with 41.6 and 38.8% recovery of protein at about 60% purity.

Beyond quantification by an ELISA specific for hCG (DSL-10-8300 kit; Diagnostic Systems Laboratories), the identity of C3 was inferred from cross-reactivity on Western blots with antibodies specific for the {alpha}- INN-hFSH-158 (OBT0346; Oxford Biotechnology, Oxfordshire, UK) and ß-subunits (BHS107; Chromaprobe Inc., Maryland Heights, MO) of hCG and by matrix-assisted laser desorption ionization-time of flight mass mapping of a tryptic digestion of the isolated ß-subunit. Purified protein was quantified by amino acid composition analysis (Keck Biotechnology Resource Facility, Yale University, New Haven, CT). Heterodimer concentration estimates by ELISA were always within a factor of two of heterodimer concentration by amino acid composition analysis. Concentrations and masses indicated throughout these studies refer to those of the heterodimeric protein.

Gonadotropin receptor binding
Previously, stable CHO cell lines were generated that express human recombinant FSHR (17) or human recombinant LHR, CHO-hFSHR, and CHO-hLHR, respectively. Large batches of those lines were grown and disrupted by nitrogen cavitation (20 min equilibration to 900 {psi}, followed by rapid pressure release) in 0.025 M Tris (pH 7.4) containing 0.25 M sucrose, 10 mM MgCl2, 1 mM EDTA, and one part per thousand protease inhibitor cocktail (p8350; Sigma, St. Louis, MO). After preliminary clarification (10 min x 1,000 x g at 4 C), the membrane fraction was pelleted (60 min x 100,000 x g x 4 C) by ultracentrifugation. The membrane fraction was resuspended in binding buffer [0.01 M Tris (pH 7.4) containing 5 mM MgCl2], protein concentration estimated by Bradford (Bio-Rad Laboratories, Hercules, CA) protein assay, and stored frozen at –80 C for future use. Typically, 15 µg of membrane protein appropriately bearing FSHR or LHR were analyzed, per well, in competition assays.

Radioligand binding was evaluated in 96-well plates, 100 µl /sample well. The assay buffer was 0.01 M Tris (pH 7.4), containing 5 mM MgCl2 and 0.1% BSA and 0.3 nM 125I-hCG (for LHR) or 0.4 nM 125I-FSH (for FSHR). Competing chimeric protein was diluted with assay buffer and mixed with radiolabeled ligand before the addition of receptor-bearing membranes. Nonspecific binding was determined in the presence of 500 nM unlabeled recombinant human (rh)CG or rhFSH. Binding was allowed to equilibrate for 90 min at 37 C with shaking. Binding assays were terminated by filtration through a low protein binding durapore membrane (Multiscreen; Millipore, Billerica, MA) preincubated in assay buffer. Filter wells were washed three times with ice-cold binding buffer (BSA-free assay buffer), dried, and punched out. Bound radioactivity was measured in an Cobra II {gamma}-counter (PerkinElmer, Wellesley, MA) using a preprogrammed detection window specific for 125I emission. Data were analyzed using a single-site model and GraphPad Prism software (GraphPad Inc., San Diego, CA).

cAMP induction assays
The same receptor expressing CHO cell lines used for membrane preparations were used, intact, to quantify cAMP production in response to agonist stimulation (Applied Biosystems cAMP-Screen Direct system, catalog no. CDC1000). When used for FSHR- or LHR-coupled cAMP assays, 96-well plates from the kit were seeded 24 h in advance at 2 x 104 cells/well of the appropriate cells in growth medium. Columns 1 (A1-H1) and 2 (A2-H2) were skipped and would be used the following day for cAMP standards supplied in the kit. On the morning of a planned assay, cell-containing wells were checked by inverted phase contrast microscopy. If the cells appeared viable and at greater than 70% confluence, the assay was completed. A determination of low confluence or low viability was followed up by reinitiating culture of the transfected cell line from a fresh vial of cryopreserved cells and repeat seeding, at a later date, for the assay. Each hormone was diluted, serially, in DMEM/F12 (phenol red-free) containing 1 mg/ml BSA and 0.10 M 3-isobutyl-1-methylxanthanone to make a 12-point test curve. Three wells of cells were stimulated for 1 h (37 C) at each dose tested. After removal of the hormone-containing medium, cells were lysed per the kit protocol and the luminescence attributable to total cAMP quantified in a Hewlett-Packard Topcount. Data were fit to the four-parameter logistic function by nonlinear least squares analysis, in MicroCal Origin (Northampton, MA), to determine EC50.

Pharmacokinetic analyses
All animal studies were approved by the Institutional Animal Care and Use Committee or Italian regulatory authorities. The serum gonadotropin concentration profiles were measured by Active hCG ELISA (Diagnostic Systems Laboratories; catalog no. DSL-10-8300 for rhCG and C3) and Active FSH ELISA (catalog no. DSL 10-4700 for rhFSH) by repeated blood sampling from immature female rats (22–23 d) after sc administration of 4 µg/animal. Pharmacokinetic parameters were calculated with software from PK Solutions (Summit Research Services, Montrose CO).

In vivo gonadotropin bioassays
Reference gonadotropins were rhFSH (lot RHST97, specific activity 93.5 ng/IU) and rhLH (lot BLCA0103, specific activity 40.0 ng/IU) obtained from Serono (Geneva, Switzerland). For quantification of FSH bioactivity in vivo, hCG-primed immature female rats were treated with equimolar amounts (2.98, 5.96, 11.94, 23.87, and 47.74 pmol/animal) of FSH, C3, or LH according to the method of Steelman and Pohley (18). LH bioactivity was determined in immature male rats after treatment with equimolar amounts (4.17, 8.35, 16.70, 33.40, and 66.80 pmol/animal) of rhLH, C3, and rhFSH according to the method of Van Hell et al. (19).

Rat follicle induction assay
Immature female rats were weaned at 21–22 d of age and randomly sorted into the experimental groups (n = 6–8/group). The rats were sc injected in the scruff of the neck twice per day (0900 and 1600 h) for 2 d with 19.2, 38.5, or 77.0 pmol/rat of rhFSH or C3 heterodimer to induce maturation of multiple ovarian follicles. The final group received vehicle alone. On the second day, at the same time as the final injection of rhFSH, C3, or vehicle, all groups received hCG (38 pmol) to induce ovulation of the matured follicles. Rats were killed on the morning after ovulation induction by CO2 asphyxiation. The ovaries, uterine horns, and uterus body were collected and placed in PBS. The oviducts were removed from the ovaries and placed between two glass microscope slides. The oviducts were examined by light microscopy under phase-contrast conditions, and ova present in the ampulla of each oviduct were counted.

The average number of ova present in the ampulla and the average ovary and uterine weight were calculated from each rat. Statistical differences between treatment groups were determined using one-way ANOVA followed by Tukey test (by S-Plus 2000 professional release 2 statistical software; Mathsoft Inc., Seattle, WA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Biophysical properties of C3
C3 is constituted from wild-type {alpha}-subunit and the chimeric FSH/hCG ß-chain (Fig. 1Go). Both hCG and C3 have apparent hydrodynamic molecular weight of approximately 75,000 as determined by analytical size exclusion chromatography (not shown). SDS-PAGE and reversed phase HPLC (RPHPLC) analyses confirm the formation of an {alpha}- and ß-chain heterodimer (Fig. 2AGo) that is acid labile (Fig. 2CGo). Like reduced rhCG visualized by Coomassie Brilliant Blue R250 protein stain (Fig. 2BGo), C3 gives rise to a single major {alpha}-subunit band and two ß-chain isoforms. Both upper bands give a single N-terminal sequence when analyzed by Edman degradation, whether the sample is a blot of reduced intact hormone or derived from the RP-HPLC-isolated ß-subunit peak (data not shown). The preparation used in these studies was qualified as approximately 60% pure heterodimer by silver stain (not shown) and ß-subunit Western blot assessment.


Figure 2
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  		FIG. 2. SDS-PAGE and RP-HPLC profiles of C3 and rhCG. Electrophoretograms of nonreduced (A) and reduced (B) C3 or rhCG were stained with Coomassie Brilliant Blue R-250. The samples (5.1 µg/lane) were analyzed on the same gel making the molecular weight calibration (Bio-Rad Precision Plus prestained) and staining valid for all. Sample properties: R, reduced; NR, nonreduced. C3 and CG have been marked on the figure. The RP-HPLC separation in C was carried out on a Vydac C8 4.5 x 250 mm column eluted in gradient mode with 0.1% trifluoroacetic acid (TFA) (A) and 0.08% TFA in CH3CN at 1 ml/min. An FC 203 fraction collector gathered 1.4 min/tube in the time windows 7.0–8.4 and 10.0–14.2 min after each injection. The three ß-subunit fractions were combined for each protein analyzed.

 
In vitro assessment of C3
Receptor binding and activation were assessed on human gonadotropin receptor-expressing CHO cells. Figure 3Go (upper panels) shows that in 125I radioligand competition assays, FSH and hCG are highly selective in binding to their respective receptors. As expected, hCG demonstrated little or no inhibition of FSH binding to FSHR, and FSH did not inhibit binding of hCG to LHR. Notably, C3 was found to efficiently inhibit both FSH binding to FSHR as well as hCG binding to LHR. The C3 inhibition constant (Ki) for FSH/FSHR binding is 4.3 ± 0.5 x 10–9 M, 4-fold higher than rhFSH at 1.0 ± 0.1 x 10–9 M. The C3 Ki for hCG/LHR binding is 10.6 ± 1.8 x 10–9 M, 53-fold higher than that of rhCG (0.2 ± 0.008 x 10–9 M). Each reported Ki represents the mean of three determinations ± SE of the mean.


Figure 3
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  		FIG. 3. Characterization of receptor-mediated cAMP stimulation in CHO cells expressing FSHR or LHR. FSHR binding was determined by competition of hCG (squares), FSH (triangles), or C3 (circles) with 125I-hFSH for binding to FSHR-expressing CHO cell membranes (upper left panel). LHR binding was determine by competition with 125I-hCG competitive binding to LHR-expressing CHO cell membranes (upper right panel). cAMP functional response was determined for hCG, FSH, and C3 with FSHR-expressing (lower left panel) and LHR-expressing intact CHO cells (lower right panel).

 
In functional assays, FSH and hCG were capable of stimulating cAMP in cells expressing human FSHR and LHR, respectively (Fig. 3Go, lower panels). In these assays, C3 was found to be a moderately potent stimulator of cAMP in FSHR-expressing cells, with an EC50 of 448 x 10–12 M corresponding to a 34-fold decrease in potency relative to rhFSH at an EC50 of 13.1 x 10–12 M. In addition, C3 stimulated cAMP in LHR-expressing CHO cells with EC50 of 955 x 10–12 M. This reflects 69-fold potency decrease relative to rhCG at EC50 13.9 x 10–12 M. In contrast, hFSH did not induce cAMP in LHR-expressing CHO cells, nor did rhCG or rhLH stimulate cAMP production in FSHR-expressing cells (not shown), indicating that the cAMP induced by C3 was due to lutropin and follitropin actions, respectively.

In vivo assessment of C3
The pharmacokinetic profile of C3 was assessed and compared with that of FSH and hCG. As expected, the mean terminal half-life of FSH (6.0 h + 0.6 h SD) was significantly shorter than that of hCG (8.6 h ± 0.5 h) after sc administration in rats. In apparent agreement with the fact that C3 contains a majority of hCG sequence, C3 displayed a longer half-life (9.7 h ± 0.8 h) than that of FSH. Peak serum concentrations (Cmax values) were not significantly different among any of the tested gonadotropins.

In vivo assessment of the gonadotropin bioactivities of C3 were determined in well-established rat models in which follitropin activity is quantitated by ovarian weight gain, and hCG/LH bioactivity is determined by seminal vesicle weight gain after gonadotropin treatment. In the ovarian weight gain model, female rats are primed with hCG (40 IU) to sensitize the rats to FSH stimulation and saturate any response that might be induced through lutropin contamination of FSH preparations often found in early urinary derived follitropins (16). In this model, FSH induced a dose-dependent increase in ovarian weight, whereas LH had no effect (Fig. 4Go, top panel). C3, when administered at the same molar amounts, resulted in a robust induction of ovarian weight gain equivalent to that observed with FSH itself. In the LH bioassay, male rats were treated with LH to induce seminal vesicle weight growth. In this assay, FSH, at the same molar doses as LH, had no impact on vesicle weight (Fig. 4Go, bottom panel). In contrast, C3 was capable of stimulating seminal vesicle weight gain at an equivalent rate as did LH. These results indicate that when administered in vivo, C3 had both follitropin and lutropin activities that were, in each case, not significantly different in potency from that of the respective recombinant versions of the wild-type gonadotropins.


Figure 4
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  		FIG. 4. In vivo assessment of follitropic and lutropic bioactivities. Ovarian weight gain dose response (top panel) was determined for FSH, C3, and LH (0, 2.98, 5.96, 11.94, 23.87, and 47.74 pmol/rat) in hCG-primed immature female rats. Seminal vesicle weight gain dose response for the same gonadotropins (0, 4.17, 8.35, 16.70, 33.40, 66.80 pmol/rat) was determined in immature male rats. Number in parentheses denotes in which of two studies the molecules were tested.

 
To determine whether the follitropin/lutropin activities of C3 were compatible with induction of folliculogenesis, immature female rats were treated with either FSH or C3 for 2 d (twice per day), followed by an ovulatory dose of hCG. When no follitropin was administered, hCG was not able to induce detectable ovulation (Fig. 5Go). However, in the presence of either FSH or C3, a dose dependent increase in the ovulation of cumulus/oocyte complexes (COCs) was observed, demonstrating that C3 was able to induce folliculogenesis with a potency similarly to that of FSH. Notably, in the absence of hCG priming, C3 induced a somewhat larger increase in uterine and ovarian weight gain than FSH, likely due to the associated lutropin bioactivity. These increases are most evident at the lower doses for the uterine response and at the higher doses for the ovarian response. Histologically, the ovaries from the FSH- and C3-treated mice were not distinguishable and displayed normal follicular stages of development (not shown). Morphologically, the COCs from the FSH- and C3-treated rats appear normal (Fig. 6Go). In contrast, when FSH was premixed with a 2-fold molar excess of LH, the resulting COCs appeared disorganized with numerous oocytes showing signs of atresia.


Figure 5
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  		FIG. 5. Effects of FSH and C3 on folliculogenesis, ovarian weight gain, and uterine weight gain. FSH or C3 (0, 19.2, 38.5, 77.0 pmol/rat twice a day x 2 d) was administered to unprimed female rats. After ovulation induction with hCG, rats were killed and ovaries, oviducts, and uteri were collected for analysis. The mean number of ovulated ova per animal (A), mean ovarian mass (B), and mean uterine mass (C), ± SEM were determined. Statistical significance relative to FSH at same dose level is denoted by asterisk.

 

Figure 6
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  		FIG. 6. Morphological characterization of ovulated cumulus-oocyte complexes after treatment of immature rats, as described in Fig. 5Go, with C3 (77 pmol/rat), FSH only (77 pmol/rat), or FSH (77 pmol/rat) plus a 2-fold molar excess of LH (154 pmol/rat). Micrographs show ova in the ampulla of an oviduct of a representative animal from each treatment group (no ovulations in vehicle-treated rats). One normal cumulus-oocyte complex in the FSH panel and one in the C3 panels is identified by an empty arrow. A disorganized complex in the 2:1 LH to FSH is denoted by the solid arrow.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Chimeric gonadotropins with similar features to C3 have been previously partially characterized in vitro (11, 12). Indeed, Wang et al. (14) have reported that of two constructs closely related to C3, CFC 94–114 and CFC 94–117, the former demonstrated greater biological activity than the latter in vivo, when tested as crude conditioned medium, in an ovarian weight gain assay. CFC 94–114 contains FSH ß-chain amino acid sequences 94–114 in the hCG backbone and, hence, differs from C3 by just three amino acids (SDS in CFC 94–114 vs. RST in C3 at amino acids 95–97). Because we and others have observed that the amino acids 94–97 (RRST) were important for LHR stimulation (13), we hypothesized that substitution at only amino acid 94 may maintain lutropin activity in addition to supporting follitropin activity. Therefore, C3 was constructed with FSH amino acid substituting at residues 101–114 in the hCG ß-chain, in addition to the single R94D substitution.

Fan and Hendrickson (20) recently published the cocrystal structure of single-chain FSH and an FSHR extracellular domain (FSHHB) that has been widely discussed (21, 22, 23). Four particular receptor residues that contact FSH in this crystal structure are not conserved among glycoprotein hormone receptors and thus probably more uniquely characterize the FSH/FSHR interaction than other hormone-receptor contacts. The nonconserved FSHHB residues that contact FSH are L55, R101 and E76 (one contact), K179, and I222. To what extent might C3 be able to make similar contacts to those of single chain FSH with FSHHB?

C3, because of its chimeric nature and the overall fold similarity between FSH and hCG, might be expected to satisfy the L55 (R101 and E76) contacts completely. Those receptor residues interact with FSH sequence that is present in C3. C3 would be unable to make the same hydrogen bond/salt bridge contact as FSH does with K179 of FSHHB. Positions 94 and 95 in C3 (Arg-Ser) are in the location homologous to Ser89-Asp90 of FSH. However, there is prior evidence that a chimeric FSH with even less potential to make this particular contact (Arg-Arg at positions 94 and 95) is still able to bind to and signal through transfected hFSHR expressed on CHO cells (24). The fourth unique FSH/FSHR contact, between I222 of FSHHB and FSH ß-loop 2 (Pro 42 and/or Pro45) might also be unavailable to C3 because C3 has Val and Ala, respectively, at the homologous ß-subunit positions. Lack of the electrostatic/polar interaction with FSHR lysine 179 and alteration of the I222ß loop 2 hydrophobic interaction seem like a reasonable explanation of follitropin receptor interaction deficiencies of C3.

Extensive in vitro characterization of the biological activities of C3 associated 4-fold decreased binding activity (Ki) with 34-fold decreased potency (cAMP EC50) relative to FSHR. In vitro C3 appeared to be a better follitropin than lutropin for CHO cells stably expressing human gonadotropin receptors because the potency reductions were 53-fold (Ki) and 69-fold (cAMP) relative to LHR. However, in vivo bioassays indicated that on a molar basis, C3-mediated gonadotropin activities were indistinguishable from those of gonadotropin standards, in contrast to in vitro activities. Although it is not clear why the apparent reduction of potency seen in vitro was not also observed in in vivo bioassays, it has been previously demonstrated that FSH variants with improved pharmacokinetic profile have increased potency in the ovarian weight gain and ovulation induction models as compared with standard rhFSH (18). This may likewise be the case with C3 because the serum half-life is significantly prolonged vs. FSH and more similar to that of hCG. Although not tested in our study, it is recognized that in various in vivo models, the serum half-lives of both hCG and FSH are greater than that of LH (25, 26). Therefore, C3 may be expected to have a greater improvement in in vivo LH potency relative to the improvement in FSH potency, resulting in the observed shift from approximately 3:1 follitropin to lutropin bioactivity ratio observed in vitro to the 1:1 ratio observed in vivo. Because gonadotropin functions are normally modulated through subtle changes in their serum concentrations, relatively small differences in the half-life of C3 might be responsible for the significant differences in bioactivities as measured in vitro and in vivo. In fact, it may be noted that subtle alterations in the C3 bioproduction and purification processes that resulted in significant reduction of apparent sialylation rate (as observed by isooelectic focusing gels) were associated with changes in relative gonadotropin potencies in vivo (not shown).

In an assay specifically designed to test whether a gonadotropin is able to support follicle maturation in immature rats, C3 treatment evoked ovarian follicles that responded to an ovulatory stimulus of hCG by releasing morphologically normal ova. Interdependent follitropin and lutropin action is a part of normal gametogenesis in vertebrates (27, 28). Under follitropin stimulation ovarian granulosa cells express the enzyme, aromatase. Interestingly, the substrate that aromatase uses for estradiol formation, androstenedione, is produced by lutropin-stimulated ovarian theca cells. Thus, there is precedent in nature for making interdependent follitropin and lutropin action a part of fertility treatment. There is also controversial clinical evidence that associates the use of lutropin during controlled ovarian stimulation with a reduction in the number of small preovulatory follicles present at the end of the cycle, thus suggesting reduced risk of ovarian hyperstimulation syndrome during early pregnancy (29). However, other studies support caution in coadministration of LH with FSH; for example, a link between excessive LH levels and polycystic ovarian syndrome has been implicated (26). Our own observation that premixing a 2-fold molar excess of hLH with rhFSH in the follicle induction assay results in morphologically abnormal COCs in rats suggests that, at least in this model, there is a upper limit on the amount of LH that can be tolerated.

Replacement therapy with follitropic and lutropic gonadotropins might be of use in treating certain infertile patients such as the normosmic population afflicted with idiopathic hypogonadotropic hypogonadism (30). Approximately one quarter of patients with idiopathic hypogonadotropic hypogonadism are not able to produce gonadotropins (31). It is known that mutations to GnRH receptor (GnRHR) perturb functional relations among three entities: GnRH secretion, signaling through its receptor (GnRHR), and the normal pattern of gonadotropin production (32). The unique biological properties demonstrated for C3 support the possibility that it may have potential in treating such patients.

We have described the generation and characterization of a novel gonadotropin with both follitropin and lutropic properties. Given the emerging evidence that some populations of subfertile patients may benefit by the addition of lutropin together with their FSH regimen, a chimeric gonadotropin with the properties of C3 may be a useful clinical tool in the treatment of these patients.


   Footnotes
 
Disclosure summary: All authors are currently or were previously employed by Serono. Authors L.M.G., K.L.A., T.M.I., E.K., and S.M. own Serono stock.

First Published Online June 22, 2006

Abbreviations: COC, Cumulus/oocyte complex; Cys, cysteine; FSHR, FSH receptor; hCG, human chorionic gonadotropin; Ki, inhibition constant; LHR, LH receptor; MTX, methotrexate; rh, recombinant human; RPHPLC, reversed phase HPLC.

Received March 20, 2006.

Accepted for publication June 12, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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