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Because of recent advancements in reproductive technology, oocytes have attained an increasingly enriched value as a unique cell population in the production of offspring. The growing oocytes in the ovary are an immediate potential source that serve this need; however, complete oocyte growth before use is crucial. Our research objective was to create in vitro-grown (IVG) oocytes that would have the ability to perform specialized activities, including nuclear reprogramming, as an alternative to in vivo-grown oocytes. Bovine oocyte-granulosa cell complexes with a mean oocyte diameter of approximately 100 μm were cultured on Millicell membrane inserts, with culture medium supplemented with 4% polyvinylpyrrolidone (molecular weight, 360,000), 20 ng/ml androstenedione, 2 mM hypoxanthine, and 5 ng/ml bone morphogenetic protein 7. Oocyte viability after the 14-day culture period was 95%, and there was a 71% increase in oocyte volume. Upon induction of oocyte maturation, 61% of the IVG oocytes extruded a polar body. Eighty-four percent of the reconstructed IVG oocytes that used cumulus cells as donor cells underwent cleavage, and half of them became blastocysts. DNA methylation analyses of the satellite I and II regions of the blastocysts revealed a similar highly methylated status in the cloned embryos derived from in vivo-grown and IVG oocytes. Finally, one of the nine embryos reconstructed from the IVG oocytes developed into a living calf following embryo transfer. Fertility of the offspring was confirmed. In conclusion, the potential of a proportion of the IVG oocytes was comparable to that of in vivo-grown oocytes.

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Cloned mammals suffer from high rates of placental abnormality and foetal loss during pregnancy. We previously used 2-D gel electrophoresis and mass spectrometry for global proteomic analysis of cloned and normal bovine placentae to identify differential protein expression patterns. Here, we used Western blot analysis to confirm the expression levels of several pregnancy-related proteins putatively identified as being differentially expressed in somatic cell nuclear transfer (SCNT) vs normal bovine placentae. The expression levels of tissue inhibitor of metalloproteinase-2 (TIMP-2), its downstream protein, matrix metalloproteinase-2 (MMP-2), superoxide dismutase (SOD), vimentin and plasminogen activator inhibitor-1 (PAI) were analysed in the placentae of SCNT cloned Korean native cattle that died immediately after birth and in normal placentae obtained by AI. Our results revealed that TIMP-2 and SOD were up-regulated in SCNT placenta compared with normal placenta, whereas MMP-2 levels were comparable in cloned and normal placentae, and vimentin and PAI were significantly down-regulated in SCNT compared with normal placentae. Our results suggest that key proteins of placental development are abnormally expressed in SCNT cloned bovine placentae, probably resulting in abnormal placental function and clonal mortality.

To investigate the ability of 1.8 kb or 3.1 kb bovine beta-casein promoter sequences for the expression regulation of transgene in vivo, transgenic mice were produced with human type II collagen gene fused to 1.8 kb and 3.1 kb of bovine beta-casein promoter by DNA microinjection. Five and three transgenic founder mice were produced using transgene constructs with 1.8 kb and 3.1 kb of bovine beta-casein promoters respectively. Founder mice were outbred with the wild type to produce F1 and F2 progenies. Total RNAs were extracted from four tissues (mammary gland, liver, kidney, and muscle) of female F1 transgenic mice of each transgenic line following parturition. RT-PCR and Northern blot analysis revealed that the expression level of transgene was variable among the transgenic lines, but transgenic mice containing 1.8 kb of promoter sequences exhibited more leaky expression of transgene in other tissues compared to those with 3.1 kb promoter. Moreover, Western blot analysis of transgenic mouse milk showed that human type II collagen proteins secreted into the milk of lactating transgenic mice contained 1.8 kb and 3.1 kb of bovine beta-casein promoter. These results suggest that promoter sequences of 3.1 kb bovine beta-casein gene can be used for induction of mammary gland-specific expression of transgenes in transgenic animals.