Reproduction in Domestic Animals

Advanced Porcine Reproductive Technology 403Other technologies related to ICSIThe establishment of a porcine IVM–ICSI proceduremay help us to generate transgenic pigs by spermmediatedgene transfer. The possibility of mammalianspermatozoa being able to act as vectors for foreignDNA has been reported by Brackett et al. (1971), andsuccessful fertilization by gene-mediated spermatozoahas been reported in mice (Lavitrano et al. 1989) andpigs (Lavitrano et al. 1997, 1999; Lazzereschi et al.2000). This method combined with ICSI has succeededin mice (Perry et al. 1999) and rats (Hirabayashi et al.2005b), and is now expected to be used in othermammalian species, including pigs. In general, theefficacy of the sperm-mediated gene transfer method,as compared with microinjection method into thepronuclei, was reviewed as excellent in the productionof transgenic animals (Smith 1999; Wall 2002). Theefficacy of transgenic pig production may be improvedby using a combination of IVM oocytes and spermmediatedgene transfer by ICSI, as has been suggestedby Kurome et al. (2006).Conservation of Ovarian Resources byXenografting into MicePrimordial follicles act as stores of ovarian follicles andare potential resources of oocytes for medical, agriculturaland zoological purposes. Ovarian grafting is apotential method of maturing the oocytes in theprimordial follicles (primordial oocytes) of large mammals.Grafting of ovarian tissues to another site in thebody (autografting) has been successful in producingviable offspring in humans (Donnez et al. 2004) andother primates (Lee et al. 2004). Cross-species ovariangrafting (xenografting) seems to be more advantageousfor the multiplication and conservation of domestic orendangered animals. Mouse oocytes that grow withinovarian tissue xenografted to nude rats acquire theability to generate pups (Snow et al. 2002). To date,ovarian tissues have been prepared from species phylogeneticallydistant from mice, including humans (Oktayet al. 1998; Weissman et al. 1999; Kim et al. 2002; Gooket al. 2003), dogs (Metcalfe et al. 2001), monkeys(Candy et al. 1995), sheep (Gosden et al. 1994), cows(Senbon et al. 2003), pigs (Kaneko et al. 2003b; Kagawaet al. 2005), tammar wallabies (Mattiske et al. 2002) andcommon wombats (Cleary et al. 2003, 2004), and thenxenografted into immunodeficient mice. To our knowledge,only our previous study (Kaneko et al. 2003b), inwhich neonatal pig ovarian tissues were xenografted,had proven that primordial oocytes can develop in thehost mice and acquire fertilizing ability.In our series of studies (Kaneko et al. 2003b, 2006;Kikuchi et al. 2006b), we have generated viable embryosfrom porcine primordial oocytes xenografted into nudemice. Here, we summarize the recent data and discuss thepossibility of further improvement of this technology.IVM–IVF of porcine oocytes grown in host miceOvarian tissues from 20-day-old piglets, in which mostof the follicles were primordial upon histological evaluation,were transplanted under the capsules of bothkidneys of ovariectomized nude mice (Kaneko et al.2003b). Forty-five to 70 days after grafting, the hostmice in all groups for the first time showed vaginalcornification. They were treated with 5 IU of equinechorionic gonadotropin (eCG) 10 days (eCG-10),30 days (eCG-30), or 60 days (eCG-60) after the detectionof cornified epithelial cells in their vaginal smears.Ovarian grafts and blood samples were obtained 48 hafter eCG treatment. The mice in all groups formedsmall numbers of antral follicles in the grafts. However,we recovered large numbers of full-sized oocytes(‡115 lm in diameter) only from mice in the eCG-60group; the numbers of full-sized oocytes in the othergroups were low. Peripheral levels of total inhibin werehighest in the eCG-60 group; this supports our findingthat the most enhanced growth of antral folliclesoccurred in this eCG-60 group. When the oocytesobtained from the eCG-60 group were matured in vitro,17% were found to be at metaphase II. Moreover, 55%of IVM oocytes with a first polar body were fertilized invitro, resulting in both male and female pronuclearformation. These results clearly demonstrate that fertilizationof oocytes from porcine primordial follicles isachievable by a combination of xenografting and in vitroculture.Developmental ability in vitro and in vivo in recipient miceafter IVF of porcine oocytes grown in most miceIn the next step, we evaluated the developmental abilityof the in vitro fertilized oocytes after transfer torecipients or in vitro culture (Kikuchi et al. 2006b). Asdetermined from the incidence of the first polar body,the maturation rates of the oocytes collected from thehost mice 48 h after treatment with eCG ranged from25.1% to 42.5%. The oocytes were fertilized in vitro andcultured in vitro for 6 days, or transferred into oestrussynchronizedrecipients and recovered after 6 days. Onday 6 of culture, 15.4% of the matured oocytes hadcleaved to the 2- to 8-cell stage. However, neither theembryos cultured in vitro nor those transferred andrecovered developed to advanced embryonic stages,such as morulae or blastocysts. These results suggestthat the developmental ability of xenografted oocytes isinsufficient, even after in vitro maturation.Improvement of developmental ability after treatment ofhost mice with gonadotropinsTo improve the developmental ability of primordialoocytes xenografted to nude mice, we treated the hostmice with gonadotrophins to accelerate folliculargrowth (Kaneko et al. 2006). Gonadotrophin treatmentswere commenced around 60 days after vaginalcornification. Ovarian grafts were obtained 2 or 3 daysafter treatment with eCG (eCG-2 and eCG-3 groups,respectively), after porcine FSH infusion by osmoticpump for 7 or 14 days, or after infusion of porcineFSH for 14 days with a single injection of estradiolantiserum (FSH-7, FSH-14 and FSH-14EA groups,respectively). Gonadotropin treatments accelerated folliculargrowth within the xenografts compared withÓ 2008 The Authors. Journal compilation Ó 2008 Blackwell Verlag