Reproduction in Domestic Animals

Recombinant Gonadotropins in Assisted Reproduction 187Fig. 1. Tissue-derived gonadotropins (LH, FSH and hCG) are composedof non-covalently linked a and b subunits each of which carryone (LHb) or two (a, FSHb and CGb) N-linked oligosaccharide chains(•). The carboxy-terminal peptide (CTP) of hCG (dashed line) alsocarries four O-linked glycans (*). In single chain gonadotropins, the aand b domains are linked by CTP. The chimeric single chaingonadotropin with dual activity (FCCGa) contains FSHb, CGb anda domains linked by CTPAs a consequence, LH, a sulphated gonadotropin, has ashort half-life. Conversely, the richly sialylated placentalcounterpart, hCG, has a much longer functional life.Deficiencies of Non-RecombinantGonadotropinsThe heterogeneous nature of tissue-derived gonadotropinpreparations results in significant variation amongbatches with respect to purity and biopotency (Murphyet al. 1984; Phillips et al. 1993; Kanitz et al. 2002). Inaddition, exogenous gonadotropins of equine or porcineorigin can induce an immune response that may limit therepetitive use of these preparations in sheep and cattle(Bodin et al. 1997; Drion et al. 2001). Furthermore, theshort half-life of ovine, porcine and human FSHpreparations necessitates continuous or repetitiveadministration, increasing the time and expense associatedwith animal sorting and handling (Alcivar et al.1992; Kanitz et al. 2002). Conversely, the long life ofeCG may compromise embryo viability (Martinuk et al.1991) and often requires the administration of anti-eCGsera during the post-ovulatory period (Gonzalez et al.1994). In addition, tissue-derived gonadotropins oftencontain differing proportions of LH and FSH activity(Kanitz et al. 2002). This heterogeneity likely contributesto variation in the physiological response (Murphyet al. 1984). A growing concern is the possibility thattissue-derived gonadotropins may harbour viruses, bacteriaor prions that may result in the inadvertentdevelopment of disease in treated animals (Reichl et al.2002; Galli et al. 2003; Matorras and Rodriguez-Escudero2003). Indeed, certain European countries havebanned the use of pituitary-derived gonadotropins insuperovulation protocols (Galli et al. 2003).Recombinant GonadotropinsRecombinant forms of LH and FSH are generated byimmortalized cells transfected with gene constructsencoding the a and b subunits (Kaetzel et al. 1985;Keene et al. 1989; Lunenfeld 2004). The transfected cellsare maintained in culture and represent a renewablesource of dimeric gonadotropin (Howles 1996). Olijveand co-workers (Olijve et al. 1996) transfected cells witha construct that incorporated the DNA encoding the aand b subunits of human FSH into a single vector. Inaddition, multiple copies of the composite constructwere stably inserted in the transfected cells (Olijve et al.1996), increasing the efficiency of dimeric FSH productionin the cell culture system. In addition, recentadvances in manufacturing and purification generaterecombinant gonadotropin preparations that are uniformin isoform profile and glycan composition (Driebergenand Baer 2003; Bassett and Driebergen 2005).These advances in recombinant gonadotropin technologyaddress many of the concerns that have developedregarding the use of tissue-derived gonadotropins.Current recombinant technology produces pharmaceuticalgrade LH and FSH of defined amino acid andcarbohydrate composition (Olijve et al. 1996). Currentpurification procedures ensure that the purity, biopotencyand batch-to-batch consistency are very high(Daya 2004). Furthermore, the generation of therecombinants in a serum-free system by immortalizeddisease-free cells ensures that the recombinant gonadotropinsare not vectors of disease. An additional benefitof the recombinant gonadotropins is that the high purityand consistent biopotency across batches permits dosingbased on mass (Driebergen and Baer 2003).The gene constructs encoding the gonadotropins aregenerally inserted into a cell line derived from ovariantissue of a Chinese hamster. The Chinese hamster ovary(CHO) cells are used from this purpose because theyaccept transfection easily (Olijve et al. 1996). In addition,CHO cells contain the intracellular machineryrequired to form the intra-chain disulphide bridges andcorrectly complete the folding and assembly of the dimer(Howles 1996). Furthermore, CHO cells constitutivelysecrete the recombinant protein into the culturemedium, a characteristic that facilitates gonadotropinrecovery and purification. Most importantly, the patternof glycosylation in CHO cells mirrors that found inpituitary-derived gonadotropins. Yet, CHO cells lackthe sulphotransferase contained in gonadotrope cells(Smith et al. 1992). As a consequence, the oligosaccharidechains of recombinant gonadotropins carry terminalsialic acid groups, rather than the terminal sulphateresidues that predominate in pituitary-derived gonadotropins(Smith et al. 1990; Baenziger et al. 1992). Thepractical consequence of sialic acid termini is that therecombinant gonadotropins are afforded protectionfrom the hepatic proteins that induce the rapid degradationof gonadotropins with sulphated termini (Baenzigeret al. 1992; Roseman and Baenziger 2000).Use of Recombinant Dimeric GonadotropinsThe efficacy of recombinant FSH in the treatment ofinfertility in humans was examined in a series of studiesconducted in the 1990s (Devroey et al. 1992; Germondet al. 1992) and the use of these recombinant gonadotropinpreparations has become common in in vitroÓ 2008 The Authors. Journal compilation Ó 2008 Blackwell Verlag