Design of a long-acting follitropin agonist by fusing the C-terminal sequence of the chorionic gonadotropin f3 subunit to the follitropin (8 subunit (biologic Communicated by Oliver H. Lowry, February 5, 1992 ABSTRACT Follitropin (FSH) is a pituitary glycoprotein hormone that is essential for the development of ovarian follicles and testicular seminiferous tubules. FSH is used clinically to stimulate follicular maturation for in vitro fertilization and treatment of anovulatory women. One issue regarding the clinical use of FSH is its short half-life in the circulation. To address this point, we constructed chimeric genes containing the sequence encoding the C-terminal peptide of the chorionic gonadotropin 13 subunit (CG1) fused to the translated sequence of the human FSH P subunit (FSH(8). This region of CGI3 is important for maintaining the prolonged plasma half-life of human CG dimer. The presence of the C-terminal peptide sequence did not significantly affect assembly of FSH(3 with the a subunit or secretion of the dimer. In vitro receptor binding and steroidogenic activity of dimer bearing the FSHf-Cterminal peptide chimera were the same as wild-type FSH.However, both the in vivo potency and half-life in circulation of the dimer bearing either one or two C-terminal peptide units were enhanced. Dimers containing FSH(-CGI3 chimeras could serve as potent FSH agonists for clinical use, and the present strategy may have wide applications for enhancing the in vivo half-life of diverse proteins.

The role of FSH in gonadal tumorigenesis and, in particular, in human ovarian cancer has been debated. It is also unclear what role the elevated FSH levels in the inhibin-deficient mouse play in the gonadal tumorigenesis. To directly assess the role of FSH in gonadal growth, differentiation, and gonadal tumorigenesis, we have generated both gain-of-function and loss-of-function transgenic mutant mice. In the gain-of-function model, we have generated transgenic mice that ectopically overexpress human FSH from multiple tissues using a mouse metallothionein-1 promoter, achieving levels far exceeding those seen in postmenopausal women. Male transgenic mice are infertile despite normal testicular development and demonstrate enlarged seminal vesicles secondary to elevated serum testosterone levels. Female transgenic mice develop highly hemorrhagic and cystic ovaries, have elevated serum estradiol and progesterone levels, and are infertile, mimicking the features of human ovarian hyperstimulation and polycystic ovarian syndromes. Furthermore, the female transgenic mice develop enlarged and cystic kidneys and die between 6-13 weeks as a result of urinary bladder obstruction. In a complementary loss-of-function approach, we have generated double-homozygous mutant mice that lack both inhibin and FSH by a genetic intercross. In contrast to male mice lacking inhibin alone, 95% of which die of a cancer cachexia-like syndrome by 12 weeks of age, only 30% of the double-mutant male mice lacking both FSH and inhibin die by 1 yr of age. The remaining double-mutant male mice develop slow-growing and less hemorrhagic testicular tumors, which are noted after 12 weeks of age, and have minimal cachexia. Similarly, the double-mutant female mice develop slow-growing, less hemorrhagic ovarian tumors, and 70% of these mice live beyond 17 weeks. The double-mutant mice demonstrate minimal cachexia in contrast to female mice lacking only inhibin, which develop highly hemorrhagic ovarian tumors, leading to cachexia and death by 17 weeks of age in 95% of the cases. The milder cachexia-like symptoms of the inhibin and FSH double-mutant mice are correlated with low levels of serum estradiol and activin A and reduced levels of aromatase mRNA in the gonadal tumors. Based on these and our previous genetic analyses, we conclude that elevated FSH levels do not directly cause gonadal tumors. However, these results suggest FSH is an important trophic modifier factor for gonadal tumorigenesis in inhibin-deficient mice.

hCG is a member of a family of glycoprotein hormones which share a common alpha-subunit, but differ in their hormone-specific beta-subunits. The CG beta-subunit is unique in that it contains a hydrophilic carboxyl-terminal extension with four serine O-linked oligosaccharides. To examine the role of the O-linked oligosaccharides and the carboxyl-terminal extension of hCG beta on receptor binding, steroidogenesis in vitro, and ovulation induction in vivo, site-directed mutagenesis and gene transfer methods were used. Wild-type hCG alpha and hCG beta expression vectors were transfected into an O-glycosylation mutant Chinese hamster ovary cell line to produce intact dimer hCG lacking the beta-subunit O-linked oligosaccharide units. In addition, a mutant hCG beta gene (CG beta delta T) was generated which contained a premature termination signal at codon 115. This gene was cotransfected with the hCG alpha gene into Chinese hamster ovary cells to produce hCG dimer which lacked the carboxyl-terminal amino acids 115-145 of hCG beta (truncated hCG). The O-linked oligosaccharide deficient or truncated hCG derivatives were examined for their ability to bind to the mouse LH/hCG receptor and stimulate cAMP and steroidogenesis in vitro. These studies show that the O-linked oligosaccharides and carboxyl-terminal extension play a minor role in receptor binding and signal transduction. In contrast, comparison of the stimulatory effects of truncated and wild-type hCG in a rat ovulation assay in vivo via either intrabursal or iv injection revealed that the truncated derivative was approximately 3-fold less active than wild-type hCG. These findings indicate that the carboxyl-terminal extension of hCG beta and associated O-linked oligosaccharides are not important for receptor binding or in vitro signal transduction, but are critical for in vivo biological responses.

Abstract. Human chorionic gonadotropin (hCG) is a member of a family of heterodimeric glycoprotein hormones that have a common ct subunit but differ in their hormone-specific 13 subunit. Site-directed mutagenesis of the two asparagine-linked glycosylation sites of hCGct was used to study the function of the individual oligosaccharide chains in secretion and subunit assembly. Expression vectors for the ~t genes (wild-type and mutant) and the hCG[3 gene were constructed and transfected into Chinese hamster ovary cells. Loss of the oligosaccharide at position 78 causes the mutant subunit to be degraded quickly and <20% is secreted. However, the presence of hCG[3 stabilizes this mutant and allows ~45% of the subunit in the form of a dimer to exit the cell. Absence of carbohydrate at asparagine 52 does not perturb the stability or transport of the ct subunit but does affect dimer secretion; under conditions where this mutant or hCGI3 was in excess, <30% is secreted in the form of a dimer. Mutagenesis of both glycosylation sites affects monomer and dimer secretion but at levels intermediate between the single-site mutants. We conclude that there are site-specific functions of the hCGct asparaginelinked oligosaccharides with respect to the stability and assembly of hCG. HUMAN chorionic gonadotropin (hCG) ~ is a member of the glycoprotein hormone family which includes lutropin, follitropin, and thyrotropin. These hormones are heterodimers consisting of an ct subunit that is common among the four hormones, and a 13 subunit that gives each its unique biological activity (5,36,53). The hCGa subunit contains two asparagine (Asn)-linked oligosaccharide chains (13, 23), whereas the hCG13 subunit contains two Asn-linked and four serine O-linked carbohydrate units (3,22,24).Several studies have addressed the role of the Asn-linked oligosaccharide chains on the gonadotropins. Chemical or enzymatic methods have been used to remove all or part of the oligosaccharide chains to obtain a better understanding of carbohydrate function. While desialylation of hCG causes it to be rapidly cleared via the hepatic route (32), its effects on biological activity in vitro are not clear (1,33,51). Removal of carbohydrate internal to sialic acid or complete deglycosylation converts hCG into an antagonist; it binds more tightly to its receptor, but shows decreased biological activity in vitro (6,21,25,33). Although the role of the oligosaccharide side-chains in folding and assembly of the hCG subunits in vivo is unknown, it has been postulated that 1. Abbreviations used in this paper: Asn, asparagine; CHO. Chinese hamster ovary; Endo F, Endoglycosidase F; Endo H, Endoglycosidase H; hCG, human chorionic gonadotropin; Thr, threonine. these oligosaccharides are important for correct disulfide bond formation (48). Combination of hCG subunits apparently occurs when the oligosaccharides are removed chemically (21, 25) or enzymatically (16). However, one problem with these methods is that incompletely deglycosylated derivatives are generated; in additio...

Corifollitropin alfa is the first long-acting hybrid molecule with sustained follicle-stimulating activity developed for the induction of multi-follicular growth along with GnRH antagonist co-treatment for IVF. This new treatment option may be simpler and more convenient for patients compared with conventional long protocols of daily FSH injections in combination with GnRH agonist co-treatment. The safety and efficacy of such regimens is currently being evaluated in large comparative phase III clinical trials. The development of corifollitropin alfa is the first step towards a new generation of recombinant gonadotrophins.

One of the distinguishing features of the gonadotropin and thyrotropin hormone family is their heterodimeric structure, consisting of a common a subunit and a hormone-specific f8 subunit. Subunit assembly is vital to the function of these hormones: The conformation of the heterodimer is essential for controlling secretion, hormonespecific posttranslational modifications, and signal transduction. To address whether a and ,3 subunits can be synthesized as one chain and also maintain biological activity, a chimera composed of the human chorionic gonadotropin (hCG) ,3 subunit genetically fused to the a subunit was constructed. question of whether the common a subunit and a hormonespecific (3 subunit can be synthesized as a single chain to achieve the biological response. For structure-function studies, the ability to express a heterodimer as a single biologically active chain would likely avoid mutagenesis-induced defects in combination and secretion of individual subunits. In addition, since subunit dissociation will inactivate the in vivo activity of the heterodimer, single-chain analogs could have a longer biological half-life. A chimera composed of the C-terminal end of hCG(3 genetically fused to the N-terminal end of the a subunit was constructed because these regions can be modified without significant effects on the biological activity of the heterodimer (7,8). Expression of this chimeric gene in CHO cells produced a single polypeptide hCG molecule that was biologically active in vitro and in vivo. These results demonstrate that the a and hCG,B subunits encoded as single chain can fold into an appropriate conformation and that a noncovalent linkage of the a and 13 subunits is not required for biological activity. This approach can be used to further investigate structure-function relationships of hCG and related glycoprotein hormones that were previously not tractable because of the absolute dependence on subunit assembly for biological activity.

FSH and luteinizing hormone (LH) are secreted constitutively or in pulses, respectively, from pituitary gonadotropes in many vertebrates, and regulate ovarian function. The molecular basis for this evolutionarily conserved gonadotropin-specific secretion pattern is not understood. Here, we show that the carboxyterminal heptapeptide in LH is a gonadotropin-sorting determinant in vivo that directs pulsatile secretion. FSH containing this heptapeptide enters the regulated pathway in gonadotropes of transgenic mice, and is released in response to gonadotropin-releasing hormone, similar to LH. FSH released from the LH secretory pathway rescued ovarian defects in Fshb-null mice as efficiently as constitutively secreted FSH. Interestingly, the rerouted FSH enhanced ovarian follicle survival, caused a dramatic increase in number of ovulations, and prolonged female reproductive lifespan. Furthermore, the rerouted FSH vastly improved the in vivo fertilization competency of eggs, their subsequent development in vitro and when transplanted, the ability to produce offspring. Our study demonstrates the feasibility to fine-tune the target tissue responses by modifying the intracellular trafficking and secretory fate of a pituitary trophic hormone. The approach to interconvert the secretory fate of proteins in vivo has pathophysiological significance, and could explain the etiology of several hormone hyperstimulation and resistance syndromes.protein sorting | regulated secretion | dense core granules | folliculogenesis

Probes derived from clones bearing cDNAs corresponding to the alpha subunit of human chorionic gonadotropin (hCG) and human placental lactogen (hPL) were used to localize their respective mRNAs cytologically in sections of first trimester and term human placenta. hPL mRNA was exclusively localized to the syncytial layer, hCG alpha mRNA was found in the syncytial layer and also in some differentiating cytotrophoblasts. Hybridization was specific because no signal was observed when labeled pBR322 was hybridized to placental sections or when the placental probes were hybridized to sections of human tonsils. In addition, RNA in placental interstitial cells did not hybridize with hCG alpha and hPL probes. Hybridization with the hCG alpha probe was much greater in first trimester than in term sections, whereas hPL signals were comparable in both first trimester and term placentae. Syncytial formation proceeds through cellular intermediates of cytotrophoblastic origin, and the data suggest that transcription of the hCG alpha gene is initiated before the completion of syncytial formation. In contrast, hPL mRNA synthesis starts later in trophoblast differentiation, likely after the stage of syncytial formation. The data also suggested that hCG alpha mRNA synthesis becomes attenuated but that hPL is transcribed at a rather constant rate during placental development.