Embryo cloning in sheep: Work in progress

Loi, Pasqualino; Boyazoglu, S.; Gallus, Marilia; Ledda, Sergio; Naitana, Salvatore; Wilmut, Ian; Cappai, P.; Casu, Sara

doi:10.1016/s0093-691x(97)00187-8

Cited by 19 publications

(7 citation statements)

References 29 publications

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“…Embryo-transfer procedures and the design, construction, and use of glass/nylon oviductal filters were based on Loi et al (1997). To ensure a consistent culture environment between NT and IVF embryos, for each replicate, embryos were transferred to a single recipient: up to 29 NT embryos were transferred to one oviduct, and up to 30 IVF embryos were transferred to the other.…”

Section: Preimplantation Embryo Culturementioning

confidence: 99%

Measures of Maternal-Fetal Interaction in Day-30 Bovine Pregnancies Derived from Nuclear Transfer

Hoffert

Batchelder

Bertolini

et al. 2005

Cloning and Stem Cells

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Embryonic mortality and abnormal placental morphology have been reported by most researchers studying nuclear transfer (NT), and it now is accepted that placental anomalies and poor development of cloned embryos are related. As early as day 50 of gestation, cloned bovine concepti exhibit poor structural organization of the developing placentomes. These experiments were designed to identify alterations in maternal-fetal interactions during establishment of the placentas of NT-derived embryos at day 30 of gestation. Bovine NT embryos were produced using cultured fibroblast cells from a single Hereford donor cow, and control embryos were derived from in vitro fertilization (IVF). Following in vivo culture in ligated sheep oviducts, day-8 blastocysts were transferred to synchronized recipient heifers. Tissues recovered from viable day-30 pregnancies were analyzed by real-time RT-PCR, immunohistochemistry, and quantitative histological techniques. Immunoperoxidase staining of caruncular tissue from NT- and IVF-derived pregnancies revealed no significant differences in expression of the extracellular matrix proteins, collagen type IV and laminin, or the receptor subunits, integrins alpha1 and alpha3, suggesting that altered expression of these proteins at day 30 of gestation is not a primary cause of abnormal placentome structure in cloned concepti. Percentage of binucleate cells (BNC) within the trophoblast also was similar in NT- and IVF-derived pregnancies; however, expression of the BNC-specific placental lactogen (PL) transcript was elevated in NT-derived concepti (p < 0.05). These results indicate that regulation of PL transcription was altered in cloned day-30 placental tissues, suggesting the presence of irregular fetal-maternal signaling patterns that might undermine continued development of NT-derived concepti.

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Section: Preimplantation Embryo Culturementioning

confidence: 99%

Measures of Maternal-Fetal Interaction in Day-30 Bovine Pregnancies Derived from Nuclear Transfer

Hoffert

Batchelder

Bertolini

et al. 2005

Cloning and Stem Cells

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“…Simultaneous fusion and activation is therefore a feature of many NT procedures. Cloned offspring have also been obtained by using electric pulses to activate recipient cytoplasts prior to NT [167,175] and reconstructed embryos following injection of donor nuclei [176].…”

Section: Activation Of Nuclear Transfer Embryosmentioning

confidence: 99%

Calcium Release at Fertilization: Artificially Mimicking the Oocyte's Response to Sperm.

Grupen¹,

Nottle²,

Nagashima

2002

Journal of Reproduction and Development

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Abstract. The mechanism of sperm-induced calcium release has been the subject of many studies since the development in the late 1950s of in vitro culture systems that support mammalian fertilization. Despite efforts to elucidate the nature of the signal from the sperm that triggers both the early and late events of oocyte activation, the precise mechanism remains unresolved. Now, with the advent of somatic nuclear transfer technologies, the need to better understand this unique process has been recognised. Nuclear transfer embryos must be induced to commence development artificially because the activating signal from the sperm is absent. The primary activating stimulus is a large increase in the concentration of intracellular-free calcium and numerous physical and chemical treatments have been found to induce calcium changes that initiate the events of oocyte activation. Although live cloned offspring have been produced in a number of species, the overall efficiencies of the nuclear transfer procedures described thus far are unacceptably low and phenotypic anomalies are common. With the aim of improving these efficiencies, researchers are developing artificial activation treatments which induce oocyte responses that mimic those induced by fertilizing sperm. One strategy is to replicate the pattern of calcium change more closely. Another strategy is to couple an activating stimulus with treatments that inhibit maturation (or M-phase) promoting factor (MPF) activity, which regulates meiotic progression in oocytes. This paper reviews what is understood of calcium release at fertilization and describes the treatments that have been used to induce oocyte activation artificially in parthenogenetic and nuclear transfer studies. The relative effectiveness of the strategies employed to mimic the oocyte's response to sperm are discussed. Key words: Oocyte activation, Fertilization, Parthenogenesis, Nuclear transfer (J. Reprod. Dev. 48: 2002) o produce a viable embryo at fertilization, the s p e r m n o t o n l y p r o v i d e s t h e m a l e chromosomal complement, but also the stimulus that initiates development. The mechanism by which the activating signal is transmitted from the sperm to the oocyte is not fully clarified, but it is clear that this signal triggers a large increase in the concentration of intracellular-free calcium. In fertilized sea urchin and Xenopus eggs, the calcium increase takes the form of a propagating wave that originates at the point of sperm attachment and traverses the egg at a velocity of 5 to 10 µm/sec

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“…Pre-activated cytoplasts increased the frequency of development to the blastocyst stage and to the term of cloned sheep embryos [57]. This procedure, however, is quite time-consuming because a two-step manipulation is required for embryo reconstruction.…”

Section: Unpublished Observations)mentioning

confidence: 99%