Kenji Naruse

Production of Fertile Offspring from Oocytes Grown In Vitro by Nuclear Transfer in Cattle

ABSTRACT 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.

Treatment of fetal fibroblasts with DNA methylation inhibitors and/or histone deacetylase inhibitors improves the development of porcine nuclear transfer-derived embryos

This study investigated whether treating fetal fibroblast cells (donor cells) with epigenetic modification-inducing drugs could improve the development of porcine cloned embryos. Donor cells were treated with different DNA methylation inhibitors (5-aza-dC, zebularine or RG108; 5nM) or histone deacetylase inhibitors (TSA, NaBu or SCR; 50nM) for 1h, and then subjected to SCNT. All of the treated groups showed significantly higher blastocyst formation rates compared to the control group. We chose 5-aza-dC and TSA as a combined treatment, and found that donor cells co-treated with 2.5nM 5-aza-dC for 1h and subsequently treated with 50nM TSA for another 1h before SCNT showed significantly improved blastocyst rates compared to the control, 5-aza-dC-treated, and TSA-treated groups. The levels of DNA methylation were decreased (though not to a significant degree) in donor cells treated with 5-aza-dC, TSA or both. The histone H3 acetylation levels were significantly increased in donor cells treated with TSA or co-treated with 5-aza-dC and TSA. Donor cells simultaneously co-treated with 5nM 5-aza-dC and 50nM TSA for 1h showed increased apoptosis of SCNT blastocysts. However, when we decreased the concentration of 5-aza-dC to 2.5nM, the co-treatment induced less apoptosis among SCNT blastocysts and the blastocyst development rate improved. Together, these results indicate that treatment of donor cells with 5-aza-dC, TSA, or TSA plus a low dose of 5-aza-dC could improve the blastocyst development of porcine cloned embryos.

Analysis of Tissue-Specific Expression of Human Type II Collagen cDNA Driven by Different Sizes of the Upstream Region of the β-Casein Promoter

To investigate the ability of 1.8 kb or 3.1 kb bovine β-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 β-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 β-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 β-casein promoter. These results suggest that promoter sequences of 3.1 kb bovine β-casein gene can be used for induction of mammary gland-specific expression of transgenes in transgenic animals.

Abnormal Expression of TIMP‐2, SOD, Vimentin and PAI Proteins in Cloned Bovine Placentae

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.

In Vitro Growth and Maturation of Vitrified-Warmed Bovine Oocytes Collected from Early Antral Follicles

Abstract. Cryopreservation of growing oocytes enriches the choice of timing and location of artificial embryo production. However, completion of oocyte growth after warming is crucial when using such cryopreserved oocytes. Our research objective was to develop a sequential system that incorporates cryopreservation of growing bovine oocytes and their subsequent in vitro growth. Oocyte-granulosa cell complexes with a mean oocyte diameter of approximately 100 µm were vitrified-warmed and then cultured for 14 days. The percentage of surviving oocytes following cryopreservation and 14-day culture was approximately 80%. More than half of the surviving oocytes were capable of maturing to metaphase II after in vitro maturation; the rate was comparable to that of control oocytes grown in vitro without cryopreservation. Taken together, the combined protocols for vitrification-warming of growing oocytes and subsequent in vitro growth can produce oocytes capable of undergoing meiotic maturation.

51 PROTEOMICS ANALYSIS OF PLACENTOMEGALY IN CLONED MICE

A variety of mammalian species have been cloned during the past few years. However, the success rate of somatic cell nuclear transfer in animals has been extremely low with many problems. Particularly, placentomegaly is a frequent finding in cloned mice and cattle (Wakayma et al. 1999 PNAS USA 96, 14 984–14 989; Niemann et al. 2000 Theriogenology 53, 21–34). To assess protein expression profile in the placentomagaly of cloned mice produced by nuclear transfer of embryonic stem cells, we have used global proteomics approach by 2-D gel electrophoresis and mass-spectrometry with the differential protein patterns using the 3 placentae of cloned mice and 4 normal mouse placentae. Proteins within isoelectric point range pH 4.0~9.0 and molecular weight range of 20–100 kDa separately were analyzed by 2-D gel electrophoresis with 3 replications of each sample. A total of approximately 3500 spots were detected 2-D gel. In the comparison of normal and cloned placenta samples, a total of 49 protein spots were expressed differentially, of which 28 spots were up-regulated proteins including alpha-fetoprotein, aspartyl aminopeptidase, placental lactogen 2, tissue inhibitor of metalloproteinase 2 (TIMP-2), etc., and 21 spots were down-regulated proteins including peroxiredoxin 6, creatine kinase, pre-B-cell colony-enhancing factor 1 (PBEF), etc. Eight spots could not be identified. One of differentially up-regulated proteins in cloned mouse placenta was identified as TIMP-2 protein that is related to extracellular matrix degradation and tissue remodeling processes. Western blot was performed with placental sample used in the 2-D gel electrophoresis analysis and normal mouse placenta samples on Days 11.5 to 18.5. Indeed, Western blot analysis confirmed a significant increase of TIMP-2 protein level in cloned mouse placenta compared with normal. The expression levels of TIMP-2 in normal mouse placenta were highest at the normal mid gestation (Day 13.5) and exhibited prominent decrease at late gestation period during normal pregnancy. However, the expression levels of TIMP-2 in placenta of cloned mice appeared to be similar to levels of mid gestation normal mouse placenta. And one of down-regulated protein in NT placenta was identified as PBEF protein that is known to be related to induction of spontaneous labor. PBEF protein level in cloned mice placenta was revealed to decrease remarkably compared with normal. The expression levels of PBEF in normal mice placenta exhibited a gradual decrease between Day 11.5 and Day 18.5 without complete depletion. However, the expression levels of PBEF in placenta of cloned mice appeared to be markedly lower than those of the same day normal placenta. In conclusion, abnormal expression of placental proteins associated with tissue remodeling and labor induction may be the cases of placentomegaly in cloned mice.