Single-chain human gonadotropin analogs induce follicle development in sheep

in Journal of Endocrinology

The biopotency of single-chain analogs of human hFSH, human chorionic gonadotropin (hCG), and a dually active gonadotropin construct (FcCGβα) was examined. Sheep (bwt=61.4±1.1 kg; n=6 ewes/treatment) received a single injection (5 IU/kg, i.v.) of the hFSH analog (Fcα), the hCG analog (CGβα), FcCGβα, or Fcα and CGβα. Control animals received conditioned media. Ovulation was induced 3 days after analog administration using hCG (1000 IU, i.v.). Basal serum concentrations of estradiol (E2) were maintained in control animals. Neither Fcα nor CGβα alone induced significant E2 production during the pre-hCG period. Conversely, serum concentrations of E2 were increased (P<0.05) 2 days after administration of FcCGβα or Fcα+ CGβα. Although P4 concentrations were maintained at basal levels in control animals, significant increase was noted in all other treatment groups during the post-hCG period. Final ovarian weight was significantly increased (P<0.05) in animals receiving Fcα, Fcα+ CGβα, or FcCGβα, but not CGβα alone. Most of the ovarian enlargement was attributed to the formation of corpora lutea. Collectively, these observations demonstrate that the single-chain analogs of the human gonadotropins are active in sheep. The construct with singular FSH activity supports follicle development but not E2 production. Conversely, the construct that incorporates β-domains from both CG and FSH has dual activity. The long-lived nature of the single-chain constructs suggests that these recombinant gonadotropins may be effective alternatives to pituitary- or placenta-derived gonadotropins in out-of-season breeding and/or superovulation protocols.

Abstract

The biopotency of single-chain analogs of human hFSH, human chorionic gonadotropin (hCG), and a dually active gonadotropin construct (FcCGβα) was examined. Sheep (bwt=61.4±1.1 kg; n=6 ewes/treatment) received a single injection (5 IU/kg, i.v.) of the hFSH analog (Fcα), the hCG analog (CGβα), FcCGβα, or Fcα and CGβα. Control animals received conditioned media. Ovulation was induced 3 days after analog administration using hCG (1000 IU, i.v.). Basal serum concentrations of estradiol (E2) were maintained in control animals. Neither Fcα nor CGβα alone induced significant E2 production during the pre-hCG period. Conversely, serum concentrations of E2 were increased (P<0.05) 2 days after administration of FcCGβα or Fcα+ CGβα. Although P4 concentrations were maintained at basal levels in control animals, significant increase was noted in all other treatment groups during the post-hCG period. Final ovarian weight was significantly increased (P<0.05) in animals receiving Fcα, Fcα+ CGβα, or FcCGβα, but not CGβα alone. Most of the ovarian enlargement was attributed to the formation of corpora lutea. Collectively, these observations demonstrate that the single-chain analogs of the human gonadotropins are active in sheep. The construct with singular FSH activity supports follicle development but not E2 production. Conversely, the construct that incorporates β-domains from both CG and FSH has dual activity. The long-lived nature of the single-chain constructs suggests that these recombinant gonadotropins may be effective alternatives to pituitary- or placenta-derived gonadotropins in out-of-season breeding and/or superovulation protocols.

Introduction

The pituitary-derived gonadotropic hormones, luteinizing hormone (LH), and follicle-stimulating hormone (FSH), are non-covalently linked heterodimeric proteins composed of a common α-subunit and a hormone-specific β-subunit (Pierce & Parsons 1981). Both gonadotropins undergo extensive co- and post-translational processing, including formation of multiple intrachain disulfide bridges, asparagine (N)-linked glycosylation, and assembly of the subunits into the dimeric configuration (Hearn & Gomme 2000). Human LH shares a high degree of structural similarity with the placenta-derived gonadotropin, human chorionic gonadotropin (hCG). Indeed, the sequence homology of the β-subunits of hLH and hCG is more than 80%. A distinctive feature of CGβ is a peptide extension at the carboxy (C) terminus. The C-terminal peptide (CTP) and its associated serine (O)-linked oligosaccharide chains markedly reduce the rate of clearance of hCG and extend its functional life (Matzuk et al. 1990).

The gonadotropic hormones exert a trophic effect on gonadal tissue and maintain the structural and functional integrity of the ovary and testis. In sheep, ovarian weight is dramatically reduced, and follicle maturation and ovulation are blocked after the removal of gonadotropin support by hypothalamo-pituitary disconnection (Hudson et al. 1999), hypophysectomy (Dufour et al. 1979), immunoneutralization of gonadotrophin-releasing hormone (GnRH; Sakurai et al. 1992), or administration of GnRH antagonists (Campbell et al. 1998). These anti-gonadal responses are reversed by administration of supplemental gonadotropic stimuli (Fry et al. 1988, Campbell et al. 1998). The magnitude of the ovarian response increases with progressive increase in the level of exogenous gonadotropin administration and high levels of supplemental FSH induce a superovulatory response (McGowan et al. 1985, Hudson et al. 1999).

The administration of exogenous gonadotropin is commonly used to enhance the fertility of humans and domestic species (Driancourt 2001, Mapletoft et al. 2002, Palagiano et al. 2004). Indeed, supplemental gonadotropins are essential components of multiple ovulation embryo transfer protocols used to enhance the fertility of domestic and exotic animals (Driancourt 2001, Mapletoft et al. 2002). The common hormone preparations (Boland et al. 1991, Lunenfeld 2004) used for this purpose are gonadotropins extracted from the pituitary tissue of sheep or pigs, the plasma or urine of pregnant women (hCG) or mares (equine chorionic gonadotropin, eCG), or the urine of post-menopausal women (human menopausal gonadotropin, hMG).

Gonadotropins derived from blood, urine, or pituitary tissue vary in potency, purity, and level of contamination with pathogenic agents or vectors of disease (Murphy et al. 1984, Manning et al. 1987, Phillips et al. 1993, Ludwig et al. 2002). Recombinant gonadotropin preparations circumvent many of these concerns (Daya 2004, Lunenfeld 2004). However, the efficiency of gonadotropin production from cells in culture is often low (Corless et al. 1987). The rate-limiting step in gonadotropin biosynthesis is assembly of the α- and β-subunits into a functional heterodimer (Garcia-Campayo et al. 1997). Subunit dimerization occurs in the endoplasmic reticulum and even under optimal conditions only about 25% of the subunits assemble properly (Bedows et al. 1992, Ruddon et al. 1996). The synthesis of single-chain gonadotropins bypasses the dimerization step and, as a consequence, increases the efficiency of in vitro production.

Single-chain analogs of conventional gonadotropins are produced by transfecting Chinese hamster ovary (CHO) cells with a gene construct that links the coding portions of the α- and β-subunit genes (Garcia-Campayo & Boime 2001b). The linker sequence used in the constructs discussed here is the exon encoding the CTP region of CGβ. The CTP segment and the associated O-linked oligosaccharide chains markedly increase the in vivo activity of the gonadotropins by reducing the rate of clearance (LaPolt et al. 1992, Joshi et al. 1995). The single-chain technology has been extended to include the union of genes encoding two or more types of β-subunits. For example, a construct containing genes encoding the α, CGβ, and FSHβ subunits produces a protein that interacts with both LH and FSH receptors. Thus, this novel chimeric gonadotropin has both LH and FSH activities in vitro (Kanda et al. 1999, Garcia-Campayo & Boime 2001a).

In the study described here, we examined the in vivo activity of the single-chain human gonadotropins with LH or FSH activity. In addition, we examined follicle development and ovulation in sheep receiving a unique chimeric gonadotropin with both LH and FSH activities. The results of these studies demonstrate that the single-chain analog with dual activity induces profound follicle development in sheep. The efficient and consistent production of this chimeric protein in a pure and pathogen-free form, along with its inherent dual activity and long functional life, may make this unique chimeric gonadotropin an effective alternative to tissue-derived gonadotropins in out-of-season breeding and superovulation protocols.

Materials and Methods

Animals

The biological activity of recombinant single-chain gonadotropins was evaluated using 30 yearling ewes (mean weight=61.4±1.1 kg). Animals were maintained under natural lighting in open-sided pens and afforded free access to water and alfalfa pellets supplemented with cereal grains and vitamin and mineral premix. The study was conducted in late fall and early winter, a period when reproductive function is at its height in sheep at this latitude (38°N). All experimental procedures involving the use of animals were conducted in accordance with NIH Guidelines and were reviewed and approved by the Animal Use and Care Committee for the University of California, Davis.

Cannulation

Polyethylene cannulae (Intramedic PE 190, Clay Adams, Parsippany, NJ, USA) were inserted into the external jugular vein, 2 days before gonadotropin administration. The cannulae were used for blood collection and administration of anti-GnRH and recombinant gonadotropin. The cannulae were protected by a sheath of plastic tubing that extended to the exterior of the animal holding area. Animals were freely mobile at the end of the 1 m lead.

Estrous synchronization

Ovarian status and estrous activity of ewes were synchronized using Lutalyse (PGF2α; Upjohn, Kalamazoo, MI, USA) and progesterone-containing intravaginal implants (Eazi-Breed CIDR (Type G), InterAg, Hamilton, NZ, USA) according to the treatment paradigm described by Van Cleeff et al. (1998).

Construction of single-chain gonadotropins

The single-chain analogs of human FSH (hFSHβ–CTP–α; Fcα), hCG (hCGβ–α; CGβα), and a chimeric gonadotropin incorporating the β-subunits of both CG and FSH (hFSHβ–CTP–hCGβ–α; FcCGβα) were generated as described by Boime et al. (Sugahara et al. 1995, 1996, Kanda et al. 1999). Briefly, these chimeric proteins were produced from gene constructs generated by joining cDNA encoding the β- and α-subunits through a linker sequence that encodes the carboxy-terminal portion of hCG. CHO cells transfected with constructs encoding Fcα, CGβα, or FcCGβα were grown to confluency and the proteins in the conditioned media concentrated by centrifugation (Centricon Plus-70, Millipore Corp., Billerica, MA, USA).

Immunization against GnRH

Antibodies directed against GnRH were generated in castrated male sheep actively immunized against a GnRH carrier protein conjugate using the procedure described previously (Adams & Adams 1986). The antisera were collected and processed for passive immunization as described by Sakurai et al. (1992). The pool of anti-GnRH sera used in these studies had high antibody titer (0.1 ml serum diluted 1:60 000 bound 44.8% of 125I-GnRH added in a final volume of 0.2 ml). Previous studies have demonstrated that administration of GnRH antisera in amounts comparable with the levels used in this study results in a block to follicle development and ovulation (Sakurai et al. 1992).

Experimental design

We hypothesized that the single-chain chimeric gonadotropin containing components of both hCG and hFSH would express dual activity and induce follicle development in sheep. The experimental protocol is presented schematically in Fig. 1. Briefly, yearling ewes were assigned at random to one of five treatment groups (n=6 ewes/group). Ovarian status and estrous activity of ewes were synchronized as described above. To negate the confounding effect of endogenous gonadotropins, animals were passively immunized against GnRH (150 ml/sheep, i.v.) 1 day before CIDR removal. The anti-GnRH serum was obtained from castrated male sheep actively immunized against GnRH as described above. Animals received CGβα, Fcα, FcCGβα, or CGβα and Fcα in combination at CIDR removal. Chimeric gonadotropins were administered at a dose of 5 IU/kg (i.v). The activity of the single-chain proteins was assessed using the immunoassay procedures discussed below. Control animals (Group 1) received an equivalent concentration of protein isolated from the conditioned media of non-transfected CHO cells. Previous studies have demonstrated that the endogenous surge of LH is not evident in sheep passively immunized against GnRH (Sakurai et al. 1992). Therefore, a bolus of hCG (1000 IU, i.v.) was administered 3 days after CIDR removal to simulate the preovulatory surge of LH. This LH-like stimulus induces ovulation in sheep deficient in endogenous gonadotropins and receiving exogenous FSH or eCG to promote follicle development (Fry et al. 1988, Picton et al. 1990). Blood samples were collected before, and at 6-h interval for 6 days after, chimera administration. Daily blood samples were collected for an additional 5-day period. Blood was allowed to clot at 4 °C, and serum was isolated by centrifugation. Serum samples were rapidly frozen and stored at −20 °C for later analysis. Animals were stunned by means of electrical shock, killed by exsanguination, and ovaries were collected 11 days after administration of the chimeric gonadotropins.

Figure 1
Figure 1

The estrous activity of 30 yearling ewes was synchronized using PGF2α and progesterone-containing vaginal inserts (CIDRs). Secretion of endogenous gonadotropins was suppressed by passive immunization with antisera directed against GnRH (anti-GnRH) 1 day prior to CIDR removal. Animals (n=6 ewes/group) received recombinant gonadotropin (CGβα, Fcα, FcCGβα, or CGβα+Fcα in combination) at CIDR removal. Chimeric gonadotropins were administered at a dose of 5 IU/kg (i.v.). Control animals received an equivalent concentration of protein isolated from the conditioned media of non-transfected CHO cells. Human chorionic gonadotropin (hCG, 1000 IU, i.v.) was administered 3 days after chimera administration to simulate the preovulatory surge of LH. Blood samples were collected at 6-h interval for 6 days after CIDR removal and daily thereafter. Ovarian tissue was collected 11 days after administration of the chimeric gonadotropins.

Citation: Journal of Endocrinology 196, 3; 10.1677/JOE-07-0473

Hormone analysis

Serum concentrations of P4 and estradiol (E2) were determined using previously validated RIAs (Adams et al. 1988, Sakurai et al. 1992). The LH and FSH activities in the single-chain analogs of the human gonadotropins were determined using dimer-specific RIAs for human LH and FSH (Diagnostic Products Corp., Los Angeles, CA, USA).

Statistical analysis

The mixed procedure in SAS was used to perform all statistical analyses. Analysis of E2 and P4 was performed using a repeated measures analysis including the fixed effects of time, treatment, and their interaction and the random effect of animal nested within the treatment×time. Differences between treatments within a time period were determined by a Bonferroni adjustment of the probability of difference calculated by the pdiff option of SAS. The analyses of area under the E2 peak, duration of elevated E2, and all ovarian measurements employed one-way ANOVA models with treatment as the sole fixed effect. Significant differences between treatments were determined by t-test with the pdiff option and Tukey's adjustment.

Results

Passive immunization against GnRH

An important aspect of our animal model involved the use of anti-GnRH sera to minimize secretion of endogenous gonadotropins. The anti-GnRH titer at chimera administration was 41.0±6% (percentage of 125I-GnRH bound by a 1:1000 dilution of serum). Antibody titer decreased gradually during the experimental period and at tissue collection (12 days after passive immunization), the anti-GnRH titer was 21.9±0.4%. The maximal titer and the rate of reduction over time did not differ (P>0.05) among treatment groups.

The effectiveness of the anti-GnRH treatment is indicated by the lack of ovarian response to CIDR removal noted in control animals receiving gonadotropin-free conditioned media. Indeed, serum concentrations of E2 in control animals were maintained at basal levels throughout the study period (Figs 2 and 3). Similarly, neither follicle development beyond the 3 mm stage nor corpora lutea were noted in ovarian tissue of control animals collected 11 days after CIDR removal. Serum concentrations of P4 were also maintained at basal levels throughout the study period in control animals (Fig. 4). Collectively, these observations indicate that the increased secretion of LH and FSH that normally accompanies CIDR removal is blocked by passive immunization.

Figure 2
Figure 2

Serum concentration of estradiol during the preovulatory period in yearling ewes receiving recombinant single-chain human gonadotropins (5 IU/kg, i.v.) at CIDR removal (day 0). Animals (n=6 ewes/treatment) received either CGβα (▾), Fcα (▴), FcCGβα (•), or CGβα+Fcα in combination (♦). Control animals (○) received an equivalent concentration of protein isolated from the conditioned media of non-transfected CHO cells.

Citation: Journal of Endocrinology 196, 3; 10.1677/JOE-07-0473

Figure 3
Figure 3

Serum concentration of estradiol during the full study period in yearling ewes receiving recombinant single-chain human gonadotropins (5 IU/kg, i.v.) at CIDR removal (day 0). Animals (n=6 ewes/treatment) received either CGβα (▾), Fcα (▴), FcCGβα (•), or CGβα+Fcα in combination (♦). Control animals (○) received an equivalent concentration of protein isolated from the conditioned media of non-transfected CHO cells. The preovulatory surge of LH was simulated by administration of hCG (1000 IU, i.v.) 3 days after chimera administration.

Citation: Journal of Endocrinology 196, 3; 10.1677/JOE-07-0473

Figure 4
Figure 4

Serum concentration of progesterone in yearling ewes receiving recombinant single-chain human gonadotropins (5 IU/kg, i.v.) at CIDR removal (day 0). Animals (n=6 ewes/treatment) received either CGβα (▾), Fcα (▴), FcCGβα (•), or CGβα+Fcα in combination (♦). Control animals (○) received an equivalent concentration of protein isolated from the conditioned media of non-transfected CHO cells. The preovulatory surge of LH was simulated by administration of hCG (1000 IU, i.v.) 3 days after chimera administration.

Citation: Journal of Endocrinology 196, 3; 10.1677/JOE-07-0473

Single-chain gonadotropin-induced E2 response

The effect of the single-chain human gonadotropin homologs on ovarian steroidogenesis during the 3-day period after chimera administration is illustrated in Fig. 2. As noted above, E2 secretion was maintained at pre-treatment levels in control animals receiving gonadotropin-free conditioned media. Basal concentrations of E2 were also evident in animals receiving the single-chain analogs of hFSH (Fcα) or hCG (CGβα). Conversely, serum concentrations of E2 increased markedly within 48 h of chimera administration in animals receiving Fcα and CGβα concurrently. A similar steroidogenic response was noted in sheep treated with the analog that incorporates the β-subunits of both hCG and hFSH (FcCGβα) into a dually active single-chain gonadotropin.

Previous studies have demonstrated that the ovulatory surge of LH is blocked in sheep passively immunized against GnRH (Sakurai et al. 1992). As a consequence, we administered supplemental hCG (1000 IU, i.v.) 3 days after chimera administration to simulate the LH surge and induce ovulation of follicles that developed during the period after CIDR removal. The initial phase of E2 production was abruptly terminated by hCG administration in animals receiving either the dually active chimera or a combination of the singly active analogs (Fig. 3). The nadir in E2 concentration was noted 12–24 h after hCG administration. A prolonged secondary phase of E2 production was noted in animals treated with FcCGβα or the Fcα+ CGβα combination. Although a post-hCG increase in E2 production was not evident in control animals or animals receiving CGβα alone, serum concentrations of E2 were significantly increased in the post-hCG period in animals receiving only Fcα at chimera administration.

Although serum concentrations of P4 were maintained at basal levels throughout the study period in control animals receiving gonadotropin-free conditioned media, P4 concentrations during the post-hCG period were significantly increased in all other treatment groups. This suggests that the simulated ovulatory surge did induce ovulation and subsequent development of corpora lutea.

Single-chain gonadotropins – trophic ovarian response

The steroidogenic response noted above provides one measure of the biological activity of the chimeric gonadotropins. The gonadotropins also exert a trophic effect on gonadal tissue that is manifest grossly as change in ovarian weight. To assess the trophic response induced by the single-chain gonadotropin analogs, we collected ovarian tissue 11 days after chimera administration. As noted in Fig. 5, total ovarian weight in animals receiving the single-chain chimera with LH activity (CGβα) did not differ from the ovarian weight in control animals. However, final ovarian weight was markedly increased in all the animals receiving supplemental FSH support. Indeed, final ovarian weight in groups receiving Fcα alone, or in combination with CGβα (Fcα+ CGβα), did not differ from the final ovarian weight in animals receiving the dually active (FcCGβα) chimera. Moreover, final ovarian weight in the latter three groups was eight- to tenfold greater than the ovarian weight noted in control animals. The marked increase in ovarian mass noted in animals receiving supplemental FSH (Fcα, Fcα+ CGβα, and FcCGβα) can be attributed, in large part, to the large number of corpora lutea evident in the tissue (Fig. 5). Although corpora lutea were not evident on the ovarian tissue of control animals, they were noted in all other groups. The most profound increase was noted in the groups receiving supplemental FSH support (Fcα, Fcα+CGβα, and FcCGβα) during the period preceding hCG administration. Interestingly, numerous large follicles were also evident on the surface of the ovaries collected from FSH-treated animals (Fcα, Fcα+ CGβα, and FcCGβα). This persistent follicle development also contributed to the enhanced ovarian weight noted in these treatment groups.

Figure 5
Figure 5

Final ovarian weight and number of corpora lutea noted at slaughter in yearling ewes receiving recombinant single-chain human gonadotropins (5 IU/kg, i.v.) at CIDR removal (day 0). Animals (n=6 ewes/treatment) received either CGβα, Fcα, FcCGβα, or CGβα+ Fcα in combination at CIDR removal. Control animals received an equivalent concentration of protein isolated from the conditioned media of non-transfected CHO cells. The preovulatory surge of LH was simulated by administration of hCG (1000 IU, i.v.) 3 days after CIDR removal and ovarian tissue was collected 8 days thereafter. The superscripts a and b denote significant difference at P<0.05 level.

Citation: Journal of Endocrinology 196, 3; 10.1677/JOE-07-0473

Discussion

The results of this study clearly demonstrate that the single-chain analogs of the human gonadotropins have potent bioactivity in sheep. The analogs with singular LH or FSH activity act in concert to promote E2 production. Interestingly, the chimeric protein incorporating both FSHβ and CGβ has dual activity in the ovine model and induces profound follicle development and E2 synthesis. Collectively, these observations indicate that the recombinant single-chain gonadotropin analogs may be useful alternatives to tissue-derived gonadotropins in treatment regimens designed to improve the reproductive efficiency of domestic species.

Estrogen production is an effective measure of the potency of the single-chain chimeric gonadotropins. As in other species (Richards 1980), E2 synthesis in sheep requires input from both the thecal and granulosa cells (Baird 1977, 1983, England et al. 1981). The essential role of the gonadotropins in ovarian function is illustrated by the arrest of follicle development and E2 synthesis in mature ewes made deficient in the gonadotropins by hypophysectomy (Draincourt et al. 1987), hypothalamo-pituitary disconnection (Hudson et al. 1999), immunoneutralization of endogenous GnRH (McNeilly et al. 1986), or administration of long-lived GnRH agonists (McNeilly & Fraser 1987) or antagonists (Campbell et al. 1998). Moreover, follicle development and E2 synthesis are reinstated in gonadotropin-deficient sheep by administration of LH and FSH (Campbell et al. 1998). Conversely, follicle development, but not E2 synthesis, is noted in gonadotropin-deficient ewes receiving supplemental FSH alone. These observations indicate that follicle development is supported by FSH, but E2 synthesis requires both gonadotropins acting in synergy.

The synergism between LH and FSH that drives E2 production is illustrated by the results noted in the study presented here. Neutralization of endogenous GnRH by passive immunization arrested follicle development at the 2–3 mm stage and blocked E2 production. Single-chain chimeras having CG or FSH activity were not effective in inducing appreciable E2 production during the 3-day period following administration of chimeric gonadotropin. However, concurrent treatment with both recombinant gonadotropins resulted in a marked increase in E2 secretion. Similarly, the single-chain chimera with dual activity also induced a significant increase in E2 synthesis. Collectively, these data demonstrate that the single-chain analogs of human CG and FSH are biologically active in sheep. In addition, the single-chain chimera that contains components of CG and FSH expresses bifunctional activity in vivo.

Although the single-chain analog of FSH does not induce E2 production in the absence of LH, follicle development is less dependent on concurrent stimulation by FSH and LH. Indeed, ovarian weight, follicular activity, and number of corpora lutea did not differ between animals receiving Fcα alone and those receiving either the dually active chimera or the Fcα+CGβα combination. These observations are consistent with previous reports noting significant FSH-induced follicle development without coincident estrogen production in LH-deficient sheep (Picton et al. 1990, Campbell et al. 1998), cattle (Hampton et al. 2004), and primates (Karnitis et al. 1994, Zelinski-Wooten et al. 1995).

A striking feature noted in animals receiving the Fcα+ CGβα combination was a biphasic pattern of E2 production. A similar pattern was also noted in animals receiving the chimeric protein with dual activity. The two phases of E2 production in these animals was punctuated by hCG-induced ovulation. The magnitude of the second phase of estrogen production was five to ten times higher than level of estrogen production common during the normal follicular phase of the ovine estrous cycle. A post-hCG surge of E2 was also noted in animals receiving the single-chain analog of FSH. This seems likely to reflect that synergy between the LH activity of the hCG administered on day 3 and the FSH activity that is intrinsic to the chimera. The secondary surge of E2 is likely not due to hCG alone since no such surge was noted in animals receiving conditioned media at CIDR removal.

The biphasic pattern of E2 production is likely reflective of the maturation and hCG-induced rupture of one cohort of antral follicles and the subsequent maturation of a second cohort. A similar biphasic pattern of follicle development and E2 secretion has been noted in heifers treated with another long-lived gonadotropin, eCG (Echternkamp 1978, Saumande 1980, Kaneko et al. 1992).

The magnitude and extended duration of the estrogenic response is likely a consequence of the long-lived nature of both hCG and the single-chain gonadotropins. Although hCG shares many features in common with LH, the unique peptide extension at the carboxy terminus of the β-subunit markedly increases the half-life of hCG (Yen et al. 1968). Since Fcα and CGβα each have a single CTP cassette and FcCGβα has two CTP units, the half-life of the single-chain gonadotropin analogs is likely to be increased. Certainly, the dramatic post-hCG surge of E2 production and the persistent follicular activity evident at slaughter are consistent with the long-lived nature of the chimeras. Moreover, preliminary observations in sheep suggest that the half-lives of Fcα and FcCGβα are about tenfold greater than that of pituitary-derived ovine FSH (Rodriquez & Adams unpublished).

The long-lived nature of the recombinant proteins is due, at least in part, to the O-linked oligosaccharide chains that are added to the CTP segment during post-translational processing. Indeed, the half-lives of both FSH (LaPolt et al. 1992) and TSH (Joshi et al. 1995, Grossmann et al. 1997) are dramatically extended by addition of the CTP segment to the β-subunit. In addition, the oligosaccharide chains of recombinant proteins expressed in CHO cells contain terminal sialic acid residues (Smith et al. 1990). Conversely, the glycoproteins produced by the ovine pituitary typically contain sulfated termini (Green & Baenziger 1988). The sialylated termini characteristic of recombinant proteins further reduce the rate of clearance by reducing the rate of hepatic extraction (Baenziger et al. 1992, Thotakura & Blithe 1995).

The dramatic follicular response induced by a single injection of the chimeric protein also reflects the long-lived nature of the single-chain gonadotropin analogs in sheep. Induction of comparable follicular development using pituitary-derived FSH generally requires repetitive or continuous administration (Campbell et al. 1998, D'Alessandro et al. 2001, 2005). Repetitive administration of hMG is also required to induce a superovulatory response in sheep, suggesting that the urinary forms of human LH and FSH that comprise hMG have a relatively short half-life in sheep. Indeed, the rate of clearance of human LH in sheep is rapid (de Kretser et al. 1973). Interestingly, the clearance of human LH in sheep is markedly reduced after nephrectomy, suggesting that renal filtration plays an important role in the clearance of pituitary-derived gonadotropins. Although the half-life of human FSH in sheep has not been determined, the rate of clearance of human FSH in cattle is rapid (Lauria et al. 1982). Collectively, these data indicate that pituitary-derived human gonadotropins, like their ovine counterparts, are quickly cleared from the circulation, while single-chain gonadotropin analogs containing one or more CTP cassettes have a much longer half-life. In practical terms, this means that a single injection of the chimera is sufficient to induce a superovulatory response. This represents a significant advantage over the current superovulation protocols that require repetitive administration of ovine, porcine, or human gonadotropins at 12-h interval for 3 days.

Taken together, the observations presented here demonstrate that the single-chain homologs of LH and FSH have activity in sheep. Although neither analog alone increases estrogenic activity, together the two chimeric gonadotropins induce marked and persistent estrogen production. Similarly, follicle development and E2 synthesis are dramatically increased in sheep receiving the chimeric gonadotropin with dual activity. These data indicate that recombinant single-chain gonadotropins with singular or dual activity may be effective alternatives to pituitary or placental gonadotropins in managed breeding and/or superovulation induction protocols.

Acknowledgements

We are grateful for the skilled technical and clinical assistance of Seth Wegner, Ben Renquist, Carla Rodriquez, and Dana Van Liew. This project was supported by National Research Initiative Competitive Grant 5-35203-16274 from the USDA Cooperative State Research, Education, and Extension Service Animal Reproduction Program, and the California Agricultural Experiment Station. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

References

  • AdamsTEAdamsBM1986Gonadotrope function in ovariectomized ewes actively immunized against gonadotropin-releasing hormone (GnRH). Biology of Reproduction35360367.

    • Search Google Scholar
    • Export Citation
  • AdamsTEQuirkeJFHanrahanJPAdamsBMWatsonJG1988Gonadotrophin secretion during the periovulatory period in Galway and Finnish Landrace ewes and Finnish Landrace ewes selected for high ovulation rate. Journal of Reproduction and Fertility83575584.

    • Search Google Scholar
    • Export Citation
  • BaenzigerJUKumarSBrodbeckRMSmithPLBeranekMC1992Circulatory half-life but not interaction with the lutropin/chorionic gonadotropin receptor is modulated by sulfation of bovine lutropin oligosaccharides. PNAS89334338.

    • Search Google Scholar
    • Export Citation
  • BairdDT1977Evidence in vivo for the two-cell hypothesis of oestrogen synthesis by the sheep Graafian follicle. Journal of Reproduction and Fertility50183185.

    • Search Google Scholar
    • Export Citation
  • BairdDT1983Factors regulating the growth of the preovulatory follicle in the sheep and human. Journal of Reproduction and Fertility69343352.

    • Search Google Scholar
    • Export Citation
  • BedowsEHuthJRRuddonRW1992Kinetics of folding and assembly of the human chorionic gonadotropin beta subunit in transfected Chinese hamster ovary cells. Journal of Biological Chemistry26788808886.

    • Search Google Scholar
    • Export Citation
  • BolandMPGouldingDRocheJF1991Alternative gonadotrophins for superovulation of cattle. Theriogenology35517.

  • CampbellBKDobsonHScaramuzziRJ1998Ovarian function in ewes made hypogonadal with GnRH antagonist and stimulated with FSH in the presence or absence of low amplitude LH pulses. Journal of Endocrinology156213222.

    • Search Google Scholar
    • Export Citation
  • Van CleeffJKarschFJPadmanabhanV1998Characterization of endocrine events during the periestrous period in sheep after estrous synchronization with controlled internal drug release (CIDR) device. Domestic Animal Endocrinology152334.

    • Search Google Scholar
    • Export Citation
  • CorlessCLMatzukMMRamabhadranTVKrichevskyABoimeI1987Gonadotropin beta subunits determine the rate of assembly and the oligosaccharide processing of hormone dimer in transfected cells. Journal of Cell Biology10411731181.

    • Search Google Scholar
    • Export Citation
  • D'AlessandroAGMartemucciGColonnaMABorgheseATerzanoMGBellittiA2001Superovulation in ewes by a single injection of pFSH dissolved in polyvinylpyrrolidone (PVP): effects of PVP molecular weight, concentration and schedule of treatment. Animal Reproduction Science65255264.

    • Search Google Scholar
    • Export Citation
  • D'AlessandroAGMartemucciGTaibiL2005How the FSH/LH ratio and dose numbers in the p-FSH administration treatment regimen, and insemination schedule affect superovulatory response in ewes. Theriogenology6317641774.

    • Search Google Scholar
    • Export Citation
  • DayaS2004Follicle-stimulating hormone in clinical practice: an update. Treatments in Endocrinology3161171.

  • DraincourtMAFryRCClarkeIJCahillLP1987Follicular growth and regression during the 8 days after hypophysectomy in sheep. Journal of Reproduction and Fertility79635641.

    • Search Google Scholar
    • Export Citation
  • DriancourtMA2001Regulation of ovarian follicular dynamics in farm animals. Implications for manipulation of reproduction. Theriogenology5512111239.

    • Search Google Scholar
    • Export Citation
  • DufourJCahillLPMauleonP1979Short- and long-term effects of hypophysectomy and unilateral ovariectomy on ovarian follicular populations in sheep. Journal of Reproduction and Fertility57301309.

    • Search Google Scholar
    • Export Citation
  • EchternkampSE1978Stimulation of estrogen and luteinizing hormone secretion in postpartum beef cows. Journal of Animal Science47521531.

    • Search Google Scholar
    • Export Citation
  • EnglandBGWebbRDahmerMK1981Follicular steroidogenesis and gonadotropin binding to ovine follicles during the estrous cycle. Endocrinology109881887.

    • Search Google Scholar
    • Export Citation
  • FryRCClarkeIJCumminsJTBindonBMPiperLRCahillLP1988Induction of ovulation in chronically hypophysectomized Booroola ewes. Journal of Reproduction and Fertility82711715.

    • Search Google Scholar
    • Export Citation
  • Garcia-CampayoVBoimeI2001aIndependent activities of FSH and LH structurally confined in a single polypeptide: selective modification of the relative potencies of the hormones. Endocrinology14252035211.

    • Search Google Scholar
    • Export Citation
  • Garcia-CampayoVBoimeI2001bNovel recombinant gonadotropins. Trends in Endocrinology and Metabolism127277.

  • Garcia-CampayoVSatoAHirschBSugaharaTMuyanMHsuehAJBoimeI1997Design of stable biologically active recombinant lutropin analogs. Nature Biotechnology15663667.

    • Search Google Scholar
    • Export Citation
  • GreenEDBaenzigerJU1988Asparagine-linked oligosaccharides on lutropin, follitropin, and thyrotropin. II. Distributions of sulfated and sialylated oligosaccharides on bovine, ovine, and human pituitary glycoprotein hormones. Journal of Biological Chemistry2633644.

    • Search Google Scholar
    • Export Citation
  • GrossmannMWongRSzkudlinskiMWWeintraubBD1997Human thyroid-stimulating hormone (hTSH) subunit gene fusion produces hTSH with increased stability and serum half-life and compensates for mutagenesis-induced defects in subunit association. Journal of Biological Chemistry2722131221316.

    • Search Google Scholar
    • Export Citation
  • HamptonJHBaderJFLambersonWRSmithMFYoungquistRSGarverickHA2004Gonadotropin requirements for dominant follicle selection in GnRH agonist-treated cows. Reproduction127695703.

    • Search Google Scholar
    • Export Citation
  • HearnMTGommePT2000Molecular architecture and biorecognition processes of the cystine knot protein superfamily. Part I. The glycoprotein hormones. Journal of Molecular Recognition13223278.

    • Search Google Scholar
    • Export Citation
  • HudsonNLO'ConnellARShawLClarkeIJMcNattyKP1999Effect of exogenous FSH on ovulation rate in homozygous carriers or noncarriers of the Booroola FecB gene after hypothalamic–pituitary disconnection or after treatment with a GnRH agonist. Domestic Animal Endocrinology166980.

    • Search Google Scholar
    • Export Citation
  • JoshiLMurataYWondisfordFESzkudlinskiMWDesaiRWeintraubBD1995Recombinant thyrotropin containing a beta-subunit chimera with the human chorionic gonadotropin-beta carboxy-terminus is biologically active, with a prolonged plasma half-life: role of carbohydrate in bioactivity and metabolic clearance. Endocrinology13638393848.

    • Search Google Scholar
    • Export Citation
  • KandaMJablonka-ShariffASatoAPixleyMRBosEHiro'okaTBen-MenahemDBoimeI1999Genetic fusion of an alpha-subunit gene to the follicle-stimulating hormone and chorionic gonadotropin-beta subunit genes: production of a bifunctional protein. Molecular Endocrinology1318731881.

    • Search Google Scholar
    • Export Citation
  • KanekoHWatanabeGTayaKSasamotoS1992Changes in peripheral levels of bioactive and immunoreactive inhibin, estradiol-17 beta, progesterone, luteinizing hormone, and follicle-stimulating hormone associated with follicular development in cows induced to superovulate with equine chorionic gonadotropin. Biology of Reproduction477682.

    • Search Google Scholar
    • Export Citation
  • KarnitisVJTownsonDHFriedmanCIDanforthDR1994Recombinant human follicle-stimulating hormone stimulates multiple follicular growth, but minimal estrogen production in gonadotropin-releasing hormone antagonist-treated monkeys: examining the role of luteinizing hormone in follicular development and steroidogenesis. Journal of Clinical Endocrinology and Metabolism799197.

    • Search Google Scholar
    • Export Citation
  • de KretserDMAtkinsRCPaulsenCA1973Role of the kidney in the metabolism of luteinizing hormone. Journal of Endocrinology58425434.

  • LaPoltPSNishimoriKFaresFAPerlasEBoimeIHsuehAJ1992Enhanced stimulation of follicle maturation and ovulatory potential by long acting follicle-stimulating hormone agonists with extended carboxyl-terminal peptides. Endocrinology13125142520.

    • Search Google Scholar
    • Export Citation
  • LauriaAGenazzaniAROlivaOInaudiPCremonesiFMonittolaCAureliG1982Clinical and endocrinological investigations on superovulation induced in heifers by human menopausal gonadotrophin. Journal of Reproduction and Fertility66219225.

    • Search Google Scholar
    • Export Citation
  • LudwigMFelberbaumREDiedrichKLunenfeldBOvarian stimulation: from basic science to clinical applicationReproductive Biomedicine Online5Suppl 120027386.

    • Search Google Scholar
    • Export Citation
  • LunenfeldB2004Historical perspectives in gonadotrophin therapy. Human Reproduction Update10453467.

  • ManningAWRajkumarKBristolFFloodPFMurphyBD1987Genetic and temporal variation in serum concentrations and biological activity of horse chorionic gonadotrophin. Journal of Reproduction and Fertility35389397.

    • Search Google Scholar
    • Export Citation
  • MapletoftRJStewardKBAdamsGP2002Recent advances in the superovulation in cattle. Reproduction Nutrition Development42601611.

  • MatzukMMHsuehAJLapoltPTsafririAKeeneJLBoimeI1990The biological role of the carboxyl-terminal extension of human chorionic gonadotropin beta-subunit. Endocrinology126376383.

    • Search Google Scholar
    • Export Citation
  • McGowanMRBraithwaiteMJochleWMaplecoftRJ1985Superovulation of beef heifers with Pergonal (HMG): a dose–response trial. Theriogenology24173184.

    • Search Google Scholar
    • Export Citation
  • McNeillyASFraserHMEffect of gonadotrophin-releasing hormone agonist-induced suppression of LH and FSH on follicle growth and corpus luteum function in the eweJournal of Endocrinology115Suppl 11987273282.

    • Search Google Scholar
    • Export Citation
  • McNeillyASJonassenJAFraserHM1986Suppression of follicular development after chronic LHRH immunoneutralization in the ewe. Journal of Reproduction and Fertility76481490.

    • Search Google Scholar
    • Export Citation
  • MurphyBDMapletoftRJMannsJHumphreyWD1984Variability in gonadotrophin preparations as a factor in the superovulatory response. Theriogenology21117125.

    • Search Google Scholar
    • Export Citation
  • PalagianoANestiEPaceL2004FSH: urinary and recombinant. European Journal of Obstetrics Gynecology and Reproductive Biology115S30S33.

    • Search Google Scholar
    • Export Citation
  • PhillipsDJHudsonNLLunSCondellLAMcNattyKP1993Biopotency in vitro and metabolic clearance rates of five pituitary preparations of follicle stimulating hormone. Reproduction Fertility and Development5181190.

    • Search Google Scholar
    • Export Citation
  • PictonHMTsonisCGMcNeillyAS1990FSH causes a time-dependent stimulation of preovulatory follicle growth in the absence of pulsatile LH secretion in ewes chronically treated with gonadotrophin-releasing hormone agonist. Journal of Endocrinology126297307.

    • Search Google Scholar
    • Export Citation
  • PierceJGParsonsTF1981Glycoprotein hormones: structure and function. Annual Review of Biochemistry50465495.

  • RichardsJS1980Maturation of ovarian follicles: actions and interactions of pituitary and ovarian hormones on follicular cell differentiation. Physiological Reviews605189.

    • Search Google Scholar
    • Export Citation
  • RuddonRWShermanSABedowsE1996Protein folding in the endoplasmic reticulum: lessons from the human chorionic gonadotropin beta subunit. Protein Science514431452.

    • Search Google Scholar
    • Export Citation
  • SakuraiHAdamsBMAdamsTE1992Pattern of gonadotropin-releasing hormone (GnRH)-like stimuli sufficient to induce follicular growth and ovulation in ewes passively immunized against GnRH. Biology of Reproduction47177184.

    • Search Google Scholar
    • Export Citation
  • SaumandeJ1980Concentrations of luteinizing hormone, oestradiol-17beta and progesterone in the plasma of heifers treated to induce superovulation. Journal of Endocrinology84425437.

    • Search Google Scholar
    • Export Citation
  • SmithPLKaetzelDNilsonJBaenzigerJU1990The sialylated oligosaccharides of recombinant bovine lutropin modulate hormone bioactivity. Journal of Biological Chemistry265874881.

    • Search Google Scholar
    • Export Citation
  • SugaharaTPixleyMRMinamiSPerlasEBen-MenahemDHsuehAJBoimeI1995Biosynthesis of a biologically active single peptide chain containing the human common alpha and chorionic gonadotropin beta subunits in tandem. PNAS9220412045.

    • Search Google Scholar
    • Export Citation
  • SugaharaTSatoAKudoMBen-MenahemDPixleyMRHsuehAJBoimeI1996Expression of biologically active fusion genes encoding the common alpha subunit and the follicle-stimulating hormone beta subunit. Role of a linker sequence. Journal of Biological Chemistry2711044510448.

    • Search Google Scholar
    • Export Citation
  • ThotakuraNRBlitheDL1995Glycoprotein hormones: glycobiology of gonadotrophins, thyrotrophin and free alpha subunit. Glycobiology5310.

  • YenSSLlerenaOLittleBPearsonOH1968Disappearance rates of endogenous luteinizing hormone and chorionic gonadotropin in man. Journal of Clinical Endocrinology and Metabolism2817631767.

    • Search Google Scholar
    • Export Citation
  • Zelinski-WootenMBHutchisonJSHessDLWolfDPStoufferRL1995Follicle stimulating hormone alone supports follicle growth and oocyte development in gonadotrophin-releasing hormone antagonist-treated monkeys. Human Reproduction1016581666.

    • Search Google Scholar
    • Export Citation

If the inline PDF is not rendering correctly, you can download the PDF file here.

 

      Society for Endocrinology

Related Articles

Article Information

Metrics

All Time Past Year Past 30 Days
Abstract Views 0 0 0
Full Text Views 177 177 8
PDF Downloads 68 68 5

Altmetrics

Figures

  • View in gallery

    The estrous activity of 30 yearling ewes was synchronized using PGF2α and progesterone-containing vaginal inserts (CIDRs). Secretion of endogenous gonadotropins was suppressed by passive immunization with antisera directed against GnRH (anti-GnRH) 1 day prior to CIDR removal. Animals (n=6 ewes/group) received recombinant gonadotropin (CGβα, Fcα, FcCGβα, or CGβα+Fcα in combination) at CIDR removal. Chimeric gonadotropins were administered at a dose of 5 IU/kg (i.v.). Control animals received an equivalent concentration of protein isolated from the conditioned media of non-transfected CHO cells. Human chorionic gonadotropin (hCG, 1000 IU, i.v.) was administered 3 days after chimera administration to simulate the preovulatory surge of LH. Blood samples were collected at 6-h interval for 6 days after CIDR removal and daily thereafter. Ovarian tissue was collected 11 days after administration of the chimeric gonadotropins.

  • View in gallery

    Serum concentration of estradiol during the preovulatory period in yearling ewes receiving recombinant single-chain human gonadotropins (5 IU/kg, i.v.) at CIDR removal (day 0). Animals (n=6 ewes/treatment) received either CGβα (▾), Fcα (▴), FcCGβα (•), or CGβα+Fcα in combination (♦). Control animals (○) received an equivalent concentration of protein isolated from the conditioned media of non-transfected CHO cells.

  • View in gallery

    Serum concentration of estradiol during the full study period in yearling ewes receiving recombinant single-chain human gonadotropins (5 IU/kg, i.v.) at CIDR removal (day 0). Animals (n=6 ewes/treatment) received either CGβα (▾), Fcα (▴), FcCGβα (•), or CGβα+Fcα in combination (♦). Control animals (○) received an equivalent concentration of protein isolated from the conditioned media of non-transfected CHO cells. The preovulatory surge of LH was simulated by administration of hCG (1000 IU, i.v.) 3 days after chimera administration.

  • View in gallery

    Serum concentration of progesterone in yearling ewes receiving recombinant single-chain human gonadotropins (5 IU/kg, i.v.) at CIDR removal (day 0). Animals (n=6 ewes/treatment) received either CGβα (▾), Fcα (▴), FcCGβα (•), or CGβα+Fcα in combination (♦). Control animals (○) received an equivalent concentration of protein isolated from the conditioned media of non-transfected CHO cells. The preovulatory surge of LH was simulated by administration of hCG (1000 IU, i.v.) 3 days after chimera administration.

  • View in gallery

    Final ovarian weight and number of corpora lutea noted at slaughter in yearling ewes receiving recombinant single-chain human gonadotropins (5 IU/kg, i.v.) at CIDR removal (day 0). Animals (n=6 ewes/treatment) received either CGβα, Fcα, FcCGβα, or CGβα+ Fcα in combination at CIDR removal. Control animals received an equivalent concentration of protein isolated from the conditioned media of non-transfected CHO cells. The preovulatory surge of LH was simulated by administration of hCG (1000 IU, i.v.) 3 days after CIDR removal and ovarian tissue was collected 8 days thereafter. The superscripts a and b denote significant difference at P<0.05 level.

References

  • AdamsTEAdamsBM1986Gonadotrope function in ovariectomized ewes actively immunized against gonadotropin-releasing hormone (GnRH). Biology of Reproduction35360367.

    • Search Google Scholar
    • Export Citation
  • AdamsTEQuirkeJFHanrahanJPAdamsBMWatsonJG1988Gonadotrophin secretion during the periovulatory period in Galway and Finnish Landrace ewes and Finnish Landrace ewes selected for high ovulation rate. Journal of Reproduction and Fertility83575584.

    • Search Google Scholar
    • Export Citation
  • BaenzigerJUKumarSBrodbeckRMSmithPLBeranekMC1992Circulatory half-life but not interaction with the lutropin/chorionic gonadotropin receptor is modulated by sulfation of bovine lutropin oligosaccharides. PNAS89334338.

    • Search Google Scholar
    • Export Citation
  • BairdDT1977Evidence in vivo for the two-cell hypothesis of oestrogen synthesis by the sheep Graafian follicle. Journal of Reproduction and Fertility50183185.

    • Search Google Scholar
    • Export Citation
  • BairdDT1983Factors regulating the growth of the preovulatory follicle in the sheep and human. Journal of Reproduction and Fertility69343352.

    • Search Google Scholar
    • Export Citation
  • BedowsEHuthJRRuddonRW1992Kinetics of folding and assembly of the human chorionic gonadotropin beta subunit in transfected Chinese hamster ovary cells. Journal of Biological Chemistry26788808886.

    • Search Google Scholar
    • Export Citation
  • BolandMPGouldingDRocheJF1991Alternative gonadotrophins for superovulation of cattle. Theriogenology35517.

  • CampbellBKDobsonHScaramuzziRJ1998Ovarian function in ewes made hypogonadal with GnRH antagonist and stimulated with FSH in the presence or absence of low amplitude LH pulses. Journal of Endocrinology156213222.

    • Search Google Scholar
    • Export Citation
  • Van CleeffJKarschFJPadmanabhanV1998Characterization of endocrine events during the periestrous period in sheep after estrous synchronization with controlled internal drug release (CIDR) device. Domestic Animal Endocrinology152334.

    • Search Google Scholar
    • Export Citation
  • CorlessCLMatzukMMRamabhadranTVKrichevskyABoimeI1987Gonadotropin beta subunits determine the rate of assembly and the oligosaccharide processing of hormone dimer in transfected cells. Journal of Cell Biology10411731181.

    • Search Google Scholar
    • Export Citation
  • D'AlessandroAGMartemucciGColonnaMABorgheseATerzanoMGBellittiA2001Superovulation in ewes by a single injection of pFSH dissolved in polyvinylpyrrolidone (PVP): effects of PVP molecular weight, concentration and schedule of treatment. Animal Reproduction Science65255264.

    • Search Google Scholar
    • Export Citation
  • D'AlessandroAGMartemucciGTaibiL2005How the FSH/LH ratio and dose numbers in the p-FSH administration treatment regimen, and insemination schedule affect superovulatory response in ewes. Theriogenology6317641774.

    • Search Google Scholar
    • Export Citation
  • DayaS2004Follicle-stimulating hormone in clinical practice: an update. Treatments in Endocrinology3161171.

  • DraincourtMAFryRCClarkeIJCahillLP1987Follicular growth and regression during the 8 days after hypophysectomy in sheep. Journal of Reproduction and Fertility79635641.

    • Search Google Scholar
    • Export Citation
  • DriancourtMA2001Regulation of ovarian follicular dynamics in farm animals. Implications for manipulation of reproduction. Theriogenology5512111239.

    • Search Google Scholar
    • Export Citation
  • DufourJCahillLPMauleonP1979Short- and long-term effects of hypophysectomy and unilateral ovariectomy on ovarian follicular populations in sheep. Journal of Reproduction and Fertility57301309.

    • Search Google Scholar
    • Export Citation
  • EchternkampSE1978Stimulation of estrogen and luteinizing hormone secretion in postpartum beef cows. Journal of Animal Science47521531.

    • Search Google Scholar
    • Export Citation
  • EnglandBGWebbRDahmerMK1981Follicular steroidogenesis and gonadotropin binding to ovine follicles during the estrous cycle. Endocrinology109881887.

    • Search Google Scholar
    • Export Citation
  • FryRCClarkeIJCumminsJTBindonBMPiperLRCahillLP1988Induction of ovulation in chronically hypophysectomized Booroola ewes. Journal of Reproduction and Fertility82711715.

    • Search Google Scholar
    • Export Citation
  • Garcia-CampayoVBoimeI2001aIndependent activities of FSH and LH structurally confined in a single polypeptide: selective modification of the relative potencies of the hormones. Endocrinology14252035211.

    • Search Google Scholar
    • Export Citation
  • Garcia-CampayoVBoimeI2001bNovel recombinant gonadotropins. Trends in Endocrinology and Metabolism127277.

  • Garcia-CampayoVSatoAHirschBSugaharaTMuyanMHsuehAJBoimeI1997Design of stable biologically active recombinant lutropin analogs. Nature Biotechnology15663667.

    • Search Google Scholar
    • Export Citation
  • GreenEDBaenzigerJU1988Asparagine-linked oligosaccharides on lutropin, follitropin, and thyrotropin. II. Distributions of sulfated and sialylated oligosaccharides on bovine, ovine, and human pituitary glycoprotein hormones. Journal of Biological Chemistry2633644.

    • Search Google Scholar
    • Export Citation
  • GrossmannMWongRSzkudlinskiMWWeintraubBD1997Human thyroid-stimulating hormone (hTSH) subunit gene fusion produces hTSH with increased stability and serum half-life and compensates for mutagenesis-induced defects in subunit association. Journal of Biological Chemistry2722131221316.

    • Search Google Scholar
    • Export Citation
  • HamptonJHBaderJFLambersonWRSmithMFYoungquistRSGarverickHA2004Gonadotropin requirements for dominant follicle selection in GnRH agonist-treated cows. Reproduction127695703.

    • Search Google Scholar
    • Export Citation
  • HearnMTGommePT2000Molecular architecture and biorecognition processes of the cystine knot protein superfamily. Part I. The glycoprotein hormones. Journal of Molecular Recognition13223278.

    • Search Google Scholar
    • Export Citation
  • HudsonNLO'ConnellARShawLClarkeIJMcNattyKP1999Effect of exogenous FSH on ovulation rate in homozygous carriers or noncarriers of the Booroola FecB gene after hypothalamic–pituitary disconnection or after treatment with a GnRH agonist. Domestic Animal Endocrinology166980.

    • Search Google Scholar
    • Export Citation
  • JoshiLMurataYWondisfordFESzkudlinskiMWDesaiRWeintraubBD1995Recombinant thyrotropin containing a beta-subunit chimera with the human chorionic gonadotropin-beta carboxy-terminus is biologically active, with a prolonged plasma half-life: role of carbohydrate in bioactivity and metabolic clearance. Endocrinology13638393848.

    • Search Google Scholar
    • Export Citation
  • KandaMJablonka-ShariffASatoAPixleyMRBosEHiro'okaTBen-MenahemDBoimeI1999Genetic fusion of an alpha-subunit gene to the follicle-stimulating hormone and chorionic gonadotropin-beta subunit genes: production of a bifunctional protein. Molecular Endocrinology1318731881.

    • Search Google Scholar
    • Export Citation
  • KanekoHWatanabeGTayaKSasamotoS1992Changes in peripheral levels of bioactive and immunoreactive inhibin, estradiol-17 beta, progesterone, luteinizing hormone, and follicle-stimulating hormone associated with follicular development in cows induced to superovulate with equine chorionic gonadotropin. Biology of Reproduction477682.

    • Search Google Scholar
    • Export Citation
  • KarnitisVJTownsonDHFriedmanCIDanforthDR1994Recombinant human follicle-stimulating hormone stimulates multiple follicular growth, but minimal estrogen production in gonadotropin-releasing hormone antagonist-treated monkeys: examining the role of luteinizing hormone in follicular development and steroidogenesis. Journal of Clinical Endocrinology and Metabolism799197.

    • Search Google Scholar
    • Export Citation
  • de KretserDMAtkinsRCPaulsenCA1973Role of the kidney in the metabolism of luteinizing hormone. Journal of Endocrinology58425434.

  • LaPoltPSNishimoriKFaresFAPerlasEBoimeIHsuehAJ1992Enhanced stimulation of follicle maturation and ovulatory potential by long acting follicle-stimulating hormone agonists with extended carboxyl-terminal peptides. Endocrinology13125142520.

    • Search Google Scholar
    • Export Citation
  • LauriaAGenazzaniAROlivaOInaudiPCremonesiFMonittolaCAureliG1982Clinical and endocrinological investigations on superovulation induced in heifers by human menopausal gonadotrophin. Journal of Reproduction and Fertility66219225.

    • Search Google Scholar
    • Export Citation
  • LudwigMFelberbaumREDiedrichKLunenfeldBOvarian stimulation: from basic science to clinical applicationReproductive Biomedicine Online5Suppl 120027386.

    • Search Google Scholar
    • Export Citation
  • LunenfeldB2004Historical perspectives in gonadotrophin therapy. Human Reproduction Update10453467.

  • ManningAWRajkumarKBristolFFloodPFMurphyBD1987Genetic and temporal variation in serum concentrations and biological activity of horse chorionic gonadotrophin. Journal of Reproduction and Fertility35389397.

    • Search Google Scholar
    • Export Citation
  • MapletoftRJStewardKBAdamsGP2002Recent advances in the superovulation in cattle. Reproduction Nutrition Development42601611.

  • MatzukMMHsuehAJLapoltPTsafririAKeeneJLBoimeI1990The biological role of the carboxyl-terminal extension of human chorionic gonadotropin beta-subunit. Endocrinology126376383.

    • Search Google Scholar
    • Export Citation
  • McGowanMRBraithwaiteMJochleWMaplecoftRJ1985Superovulation of beef heifers with Pergonal (HMG): a dose–response trial. Theriogenology24173184.

    • Search Google Scholar
    • Export Citation
  • McNeillyASFraserHMEffect of gonadotrophin-releasing hormone agonist-induced suppression of LH and FSH on follicle growth and corpus luteum function in the eweJournal of Endocrinology115Suppl 11987273282.

    • Search Google Scholar
    • Export Citation
  • McNeillyASJonassenJAFraserHM1986Suppression of follicular development after chronic LHRH immunoneutralization in the ewe. Journal of Reproduction and Fertility76481490.

    • Search Google Scholar
    • Export Citation
  • MurphyBDMapletoftRJMannsJHumphreyWD1984Variability in gonadotrophin preparations as a factor in the superovulatory response. Theriogenology21117125.

    • Search Google Scholar
    • Export Citation
  • PalagianoANestiEPaceL2004FSH: urinary and recombinant. European Journal of Obstetrics Gynecology and Reproductive Biology115S30S33.

    • Search Google Scholar
    • Export Citation
  • PhillipsDJHudsonNLLunSCondellLAMcNattyKP1993Biopotency in vitro and metabolic clearance rates of five pituitary preparations of follicle stimulating hormone. Reproduction Fertility and Development5181190.

    • Search Google Scholar
    • Export Citation
  • PictonHMTsonisCGMcNeillyAS1990FSH causes a time-dependent stimulation of preovulatory follicle growth in the absence of pulsatile LH secretion in ewes chronically treated with gonadotrophin-releasing hormone agonist. Journal of Endocrinology126297307.

    • Search Google Scholar
    • Export Citation
  • PierceJGParsonsTF1981Glycoprotein hormones: structure and function. Annual Review of Biochemistry50465495.

  • RichardsJS1980Maturation of ovarian follicles: actions and interactions of pituitary and ovarian hormones on follicular cell differentiation. Physiological Reviews605189.

    • Search Google Scholar
    • Export Citation
  • RuddonRWShermanSABedowsE1996Protein folding in the endoplasmic reticulum: lessons from the human chorionic gonadotropin beta subunit. Protein Science514431452.

    • Search Google Scholar
    • Export Citation
  • SakuraiHAdamsBMAdamsTE1992Pattern of gonadotropin-releasing hormone (GnRH)-like stimuli sufficient to induce follicular growth and ovulation in ewes passively immunized against GnRH. Biology of Reproduction47177184.

    • Search Google Scholar
    • Export Citation
  • SaumandeJ1980Concentrations of luteinizing hormone, oestradiol-17beta and progesterone in the plasma of heifers treated to induce superovulation. Journal of Endocrinology84425437.

    • Search Google Scholar
    • Export Citation
  • SmithPLKaetzelDNilsonJBaenzigerJU1990The sialylated oligosaccharides of recombinant bovine lutropin modulate hormone bioactivity. Journal of Biological Chemistry265874881.

    • Search Google Scholar
    • Export Citation
  • SugaharaTPixleyMRMinamiSPerlasEBen-MenahemDHsuehAJBoimeI1995Biosynthesis of a biologically active single peptide chain containing the human common alpha and chorionic gonadotropin beta subunits in tandem. PNAS9220412045.

    • Search Google Scholar
    • Export Citation
  • SugaharaTSatoAKudoMBen-MenahemDPixleyMRHsuehAJBoimeI1996Expression of biologically active fusion genes encoding the common alpha subunit and the follicle-stimulating hormone beta subunit. Role of a linker sequence. Journal of Biological Chemistry2711044510448.

    • Search Google Scholar
    • Export Citation
  • ThotakuraNRBlitheDL1995Glycoprotein hormones: glycobiology of gonadotrophins, thyrotrophin and free alpha subunit. Glycobiology5310.

  • YenSSLlerenaOLittleBPearsonOH1968Disappearance rates of endogenous luteinizing hormone and chorionic gonadotropin in man. Journal of Clinical Endocrinology and Metabolism2817631767.

    • Search Google Scholar
    • Export Citation
  • Zelinski-WootenMBHutchisonJSHessDLWolfDPStoufferRL1995Follicle stimulating hormone alone supports follicle growth and oocyte development in gonadotrophin-releasing hormone antagonist-treated monkeys. Human Reproduction1016581666.

    • Search Google Scholar
    • Export Citation

Cited By

PubMed

Google Scholar