Reproduction in Domestic Animals

Recombinant Gonadotropins in Assisted Reproduction 189the serum concentration of testosterone after administrationto stallions. The single chain analogue was alsoas effective as hCG in inducing ovulation in mares(Yoon et al. 2007). Indeed, ovulation was evident within48 h of analogue administration in mares with a maturefollicle (>35 mm) at the time of administration. Ovulationwas noted within the same temporal window inonly 15% of control mares receiving vehicle alone. Yoonand colleagues suggest that the single chain analogue ofeLH may be useful in accelerating ovulation and,thereby, facilitating timed mating and inseminationusing frozen ⁄ thawed semen.The bioactivity of single chain analogues of hFSH hasbeen assessed in rodent (Weenen et al. 2004) andprimate (Klein et al. 2002) models. Administration ofa single dose of the FSH analogue to prepuberal ratsinduced a significant increase in ovarian weight andinhibin secretion (Weenen et al. 2004) and promoted thedevelopment of multiple antral follicles (Trousdale et al.2007). In contrast, ovarian weight and inhibin productiondid not differ from saline-treated control animals inrats receiving a single injection of dimeric rhFSH. Thisindicates that the functional life of the single chainanalogue is greater than that of dimeric FSH. Indeed,direct assessment of the half-life indicated that theclearance of the single chain analogue was three timesslower than the clearance of dimeric FSH in the rodentmodel. Follicle development and oestradiol productionwere also increased in monkeys receiving a singleinjection of the single chain FSH analogue (Klein et al.2002). As in the rodent model, the half-life of the FSHanalogue was markedly increased relative to the half-lifeof recombinant dimeric FSH. Collectively, these studiesdemonstrate that the single chain form of FSH is activeand long-lived.Gonadotropins with Dual ActivityA novel variation of the single chain technology involvesthe incorporation of two or more different b subunitdomains into the single chain protein (Kanda et al.1999; Garcia-Campayo and Boime 2001). When examinedusing in vitro assay systems a single chaingonadotropin with a, hFSHb and hCGb domains(FSHb–CTP–CGb–a) interacted with LH and FSHreceptors and induced cAMP production that wascomparable to the level induced by dimeric LH andFSH (Kanda et al. 1999). The dual activity of thechimeric gonadotropin suggests that the single chainprotein can assume a secondary structure that isrecognized by both gonadotropin receptors. Yet, recentstudies indicate that the chimeric proteins assume twodifferent conformations, one that is recognized by theFSH receptor and a second configuration that permitsrecognition by the LH receptor (Garcia-Campayo et al.2004). In other words, the single chain proteins secretedfrom transfected CHO cells represent a heterogeneouspopulation of conformations, some with the capacity toactivate the LH receptor and others capable of activatingthe FSH receptor. The dual activity of the chimerichuman gonadotropins is similar to the bifunctionalpotential of eCG. Indeed, the dually active chimericgonadotropin was as effective as eCG in promotingfollicle development, enhancing of ovarian weight andaugmenting aromatase activity in immature mice (Jablonka-Shariffet al. 2006). The ovulation inductionaction of hCG was also mimicked by the dually activechimeric gonadotropin in eCG-primed immature mice.In our recent studies, the authors have compared thebiopotency of the dually active human gonadotropinwith single chain analogues of hFSH and hCG (Lemkeet al. 2008). The research model we used employed ewestreated with progesterone-containing vaginal inserts(CIDR’s) and anti-GnRH sera to suppress secretion ofendogenous gonadotropins (Fig. 2). Ewes received asingle injection of the dually active gonadotropin(FSHb–CTP–CGb–a; 5 IU⁄ kg, iv), single chain analoguesof hFSH (FSHb–CTP–a; Fca; 5IU⁄ kg, iv) orCG (CGb–a; CGba; 5IU⁄ kg, iv), or the FSH and CGanalogues in combination (Fca +CGba; both at5IU⁄ kg, iv) at the time of CIDR removal. Controlanimals received vehicle alone. Ovulation was inducedby hCG (1000 IU, iv) injection 3 days after administrationof the single chain gonadotropins.As oestradiol production requires the concertedaction of both LH and FSH, the authors used oestradiolsecretion as a measure of the bifunctional capacity of thesingle chain gonadotropins. Significant oestradiol secretionwas not evident in ewes receiving Fca or CGbaalone. Yet, serum levels of oestradiol were markedlyincreased in ewes receiving Fca and CGba in combination.The results of this study demonstrate that thesingle chain analogues of hFSH and hCG are active insheep. These data also clearly illustrate that the singlechain construct that incorporates FSHb and CGbdomains has both FSH and LH activity (Table 1).Similarly, follicle development was promoted by thedually active chimeric gonadotropins and the combinedtreatment with Fca and CGba. This augmented follicularresponse is indicated by the increase in ovarianweight and corpora lutea number that were noted inovarian tissue collected 11 days after administration ofFig. 2. The oestrous activity of yearling ewes was synchronized usingprogesterone-containing vaginal inserts (CIDRs). Secretion of endogenousgonadotropins was suppressed by passive immunization withantisera directed against GnRH (Anti-GnRH) 1 day prior to CIDRremoval. Animals (n = 6 ewes ⁄ group) received recombinant gonadotropin(CGba, Fca, FcCGba or CGba +Fca in combination) atCIDR removal. Chimeric gonadotropins were administered at a doseof 5 IU ⁄ kg (iv). Control animals received conditioned media of nontransfectedCHO-cells. Ovulation was induced by hCG (1000 IU, iv)on day 3 and ovarian tissue was collected on day 11Ó 2008 The Authors. Journal compilation Ó 2008 Blackwell Verlag