Reproduction in Domestic Animals

188 TE Adams and I Boimefertilization programs (Daya 2004). In the animalindustries, recombinant forms of bovine (Wilson et al.1993; Wehrman et al. 1996) and human (Takagi et al.2001) FSH (rbFSH and rhFSH, respectively) have beenused to induce superovulation in beef cattle. Recombinantporcine FSH (rpFSH) has also been produced andthe biological activity has been verified using an in vitroSertoli cell bioassay and an in vivo assay based on follicledevelopment in hypophysectomized mice treated withrpFSH (Inaba et al. 1997).The comprehensive studies of Wilson and co-workers(Wilson et al. 1993) demonstrated that rbFSH was aseffective as pituitary-derived FSH in promoting thesuperovulatory response in mature cows. The treatmentprotocol required rbFSH administration (im) at 12 hintervals for 3–5 days. The optimal response was notedat a total dose of 24 mg rbFSH. This level of rbFSH iscomparable to the total dose of purified pituitary FSHrequired to induce superovulation in beef and dairycows (Alcivar et al. 1992; Kanitz et al. 2002). Thedevelopment of multiple pre-ovulatory follicles was alsoinduced in heifers receiving twice daily injections ofrhFSH over a 4-day-treatment period (Takagi et al.2001). The magnitude of the rhFSH-induced responsewas comparable to the follicular response induced byeCG. Yet, when compared with eCG-treated heifers,serum concentrations of oestradiol during the preovulatoryperiod were significantly reduced in heifersreceiving rhFSH. As a consequence, the induction of thepre-ovulatory surge of LH was delayed. Takagi and coworkerssuggest that endogenous production of LH isnot sufficient to support oestradiol synthesis duringrhFSH-induced follicle development. Therefore, concurrentadministration of LH and rhFSH may berequired to achieve an ovulatory response that iscomparable to that induced by eCG.Dimeric CTP-Modified GonadotropinsIn contrast to pituitary-derived gonadotropins, placentalgonadotropins are cleared slowly and, as a consequence,the duration of response induced by eCG orhCG is much longer than the response induced by LH orFSH (Martinuk et al. 1991; Baenziger et al. 1992). Thelong half-life of the placental gonadotropins is due, inpart, to a carboxyl terminal peptide (CTP) extension ofthe b subunit that is subject to extensive O-linkedglycosylation (Kessler et al. 1979a; Bousfield et al.2001). The critical impact of the CTP on gonadotropinclearance was demonstrated by Matzuk and co-workers(Matzuk et al. 1990), who produced a truncated form ofhCGb lacking the CTP domain. Although the truncatedvariant of hCG was recognized with high affinity by theLH receptor, the in vivo potency was dramaticallyreduced by deletion of the CTP domain. Similarly,incorporating a CTP domain into the b subunits of FSHor TSH markedly increases the half-life of the CTPmodifiedforms of FSH and TSH (Fares et al. 1992;Joshi et al. 1995).Although the bioactivity of CTP-modified dimericFSH has not been examined in domestic species, theefficacy of an hFSH analogue incorporating the CTPdomain (hFSH-CTP) has been examined in humans.The hFSH analogue is effective in promoting spermatogenesisand follicle development (Bouloux et al.2001; Balen et al. 2004). Importantly, in comparisonwith rhFSH, the rate of clearance of hFSH-CTP ismarkedly reduced and development of pre-ovulatoryfollicles is induced by a single injection of the FSHanalogue. This is in contrast to the repetitive injectionschedule that is required to promoted follicle maturationusing pituitary-derived FSH. Collectively, thesedata demonstrate that the CTP domain extends thefunctional life of FSH and simplifies the ovulationinduction protocol by reducing the frequency ofadministration.Single Chain Analogues of LH and FSHA recent advance in the production of recombinantgonadotropins is the generation of single chain analoguesof the normally dimeric proteins (Garcia-Campayoand Boime 2001). The single chain technologyinvolves transfection of CHO cells with a plasmid thatcarries DNA encoding both subunits combined in asingle gene construct. The linker sequence that joins thea and b domains of the construct encodes the CTPportion of hCGb. The protein product secreted bytransfected CHO cells is a single peptide chain thatcontains a and b domains linked through the CTPsegment. To date, single chain analogues of hFSH(FSHb–CTP–a; Sugahara et al. 1996), hLH (LHb–CTP–a; Garcia-Campayo et al. 1997), equine LH(eLHb–CTP–a; Jablonka-Shariff et al. 2007) and hCG(CGb–a; Sugahara et al. 1995) have been developed.The single chain analogues of the gonadotropins arerecognized by the appropriate receptor (LH or FSHreceptor) with high affinity and the single chain analoguesinduce the normal intracellular second messengerresponse. The bioactivity of the single chain analoguesdemonstrates that the folding of the a and b componentsinto the configuration required to associate with thegonadotropin receptor is not impaired by the singlechain organization of the a and b domains. The correctfolding is likely facilitated by the intervening CTPsequence. Importantly, the high proportion of prolineand serine residues in the CTP sequence makes thissegment highly flexible and allows the a and b domainsof the single chain to fold into the active configuration.An additional benefit of incorporating the CTPsequence into the single chain is that, much like thedimeric CTP-modified gonadotropins discussed above,the CTP portion of the single chain reduces the rate ofclearance. An additional practical consequence of producinggonadotropins as single chain proteins is thatdimerization, the rate limiting step in the synthesis ofconventional gonadotropins, is bypassed. This uniquefeature of the single chain proteins enhances theefficiency of synthesis and secretion by transfectedCHO cells (Sugahara et al. 1996).The recent studies of Jablonka-Shariff and co-workersexamined the activity of a single chain analogue ofequine LH (Jablonka-Shariff et al. 2007). The analoguewas as potent as pituitary-derived eLH in promotingtestosterone production by equine Leydig cells inculture. The single chain eLH analogue also increasedÓ 2008 The Authors. Journal compilation Ó 2008 Blackwell Verlag