Reproduction in Domestic Animals

Developmental Capabilities of Prepubertal Lamb Embryos 141during hormone stimulation, or differences in therespective culture systems, including media (KM Mortonand SL Catt, unpublished data) and oxygen tension(Leoni et al. 2007).The kinetics of both oocyte maturation (Dominko andFirst 1997) and embryo development (Van Soom et al.1997) indicate the developmental capabilities of embryos.The reduced speed of development of prepubertal derivedembryos may explain the low, or reduced in vivo survivalreported by numerous workers (Quirke and Hanrahan1977; McMillan and McDonald 1985; O’Brien et al.1997a; Ptak et al. 1999, 2003, 2006; Kelly et al. 2005b). Inaddition to this low survival rate, remarkably high ratesof embryonic ⁄ foetal loss (O’Brien et al. 1997a; Ptak et al.1999, 2003, 2006) and mummification (Ptak et al. 1999)have been reported. Ptak et al. (2006) reported severelyinterrupted pregnancy (between days 40 and 60) andfoetal losses (days 80–100) with only 6% of the 628embryos transferred surviving until full gestation.This reduced development is generally attributed toincomplete or perturbed cytoplasmic maturation, whichmay be expressed at many stages of embryonic development.Perturbed cytoplasmic maturation commonlypresents as a failure of sperm penetration and decondensation,inability to form normal male pronuclei,failure of the block to polyspermy, early cleavagefailure, failure to reach or survive the transition frommaternal to embryonic genome expression, and developmentalfailure leading to embryonic losses at laterpre-implantation and post-implantation stages of development(Armstrong 2001). All of these have beenreported for oocytes and embryos derived from prepubertalanimals, and the evidence regarding perturbedcytoplasmic maturation in prepubertal oocytes is nowoverwhelming.The perturbed cytoplasmic maturation is generallyattributed to the age of the donors and the lack of timeto sequester factors into the cytoplasm (Armstrong2001). Yet, Ptak et al. (2006) also demonstrated incompletenuclear maturation of prepubertal lamb oocytes,and the authors suggest that the previous definition ofnuclear competence (i.e. the ability to complete meiosis)was inadequate. Furthermore, the authors observeddiscordance between follicle and oocyte growth, leadingto incomplete or perturbed oocyte growth. This, accordingto Ptak et al. (2006), resulted in incomplete developmentof both the nuclear and cytoplasmiccompartments, which was responsible for the reduceddevelopmental competence of oocytes derived fromprepubertal animals.Reduction in global genome methylation (Ptak et al.2006) and mRNA storage (Leoni et al. 2006a) havebeen reported for oocytes derived from prepubertallambs. Ptak et al. (2006) observed that genome-widemethylation status (% of methylated vs total DNA)was lower for oocytes derived from prepubertal lambswhen compared with adult animals. The authorshypothesize that the reduced global methylation affectscertain imprinted genes as the patterns of foetal lossobserved by Ptak et al. (1999, 2006) are similar to micelacking oocyte-specific DNA methyltransferase-1(Dnmt1o) where the majority of foetus die in the lastthird of gestation (Howell et al. 2001). The reducedmethylation, mRNA storage and perturbed growth ofoocytes from prepubertal animals, provides strongevidence for the contribution of nuclear immaturityto their reduced developmental competence. Furtherstudies aiming to locate the specific sites where thedifferences in methylation occur will provide molecularexplanations for the differences in the developmentalcapabilities of oocytes from adult and prepubertalanimals.Most workers, most notably Ptak et al. (1999, 2006),have reported low rates of in vitro development forprepubertal oocytes accompanied by substantial embryonicand foetal loss. In other studies, by contrast,prepubertal oocytes have displayed a high developmentalcompetence in vitro not dissimilar to that of oocytesfrom adult sheep, suggesting that the use of large FSHdoses during hormone stimulation may attenuate manyof the problems associated with JIVET, specifically thehigh proportion of lambs which fail to respond tohormone stimulation, high rates of fertilization abnormalities,reduced and delayed development to the blastocyststage and compromised embryo survival aftertransfer to recipient ewes (Morton et al. 2005b; c, d).While Ptak et al. (1999, 2006) and Morton et al.(2005b,c,d) utilized oocytes from lambs of a similar age(4 weeks), the different breeds of the donors, hormonestimulation regimes (in particular, the FSH dose: 2.7 or7 mg and 130 mg pFSH, respectively) and IVP systemsconfound comparisons between these studies. The role ofin vitro culture on the perturbation of embryonic geneexpression is well-documented (Wrenzycki et al. 2005).Yet, the effects of different IVC systems and hormonestimulation with a high FSH regime during oocytegrowth on gene expression, have yet to be investigatedfor oocytes from prepubertal animals.Despite the reduced developmental competence ofoocytes from prepubertal animals, offspring have beenproduced from both fresh and frozen-thawed embryosderived from lambs as young as 3–4 weeks of age(Morton et al. 2004a, 2005d) and the improvements tothe efficiency of JIVET technology have facilitated theincorporation of JIVET with other emerging reproductivetechnologies such as sperm sexing (Maxwell et al.2004) for the production of pre-sexed embryos andoffspring.Future Directions and PerspectivesLimitations to the commercial application of JIVEThave included the high proportion of donors that fail torespond to hormone stimulation, the highly variableresponse to hormone stimulation and the reduceddevelopmental competence of oocytes derived fromprepubertal animals. Using a high FSH stimulationregime (Morton et al. 2004a, 2005a, b, c, d) all lambsresponded to hormone stimulation and oocyte developmentalcompetence was significantly improved. Developmentrate was similar for oocytes derived fromprepubertal and adult ewes, and in vivo survival afterembryo transfer was high and free from the highmalformation rates reported by other authors.The advent of genomic technologies has furtherincreased our knowledge on the differences betweenÓ 2008 The Author. Journal compilation Ó 2008 Blackwell Verlag