Identification of amniotic fluid metabolomic and placental transcriptomic changes associated with abnormal development of cloned pig fetuses.

Piglets cloned by somatic cell nuclear transfer (SCNT) show a high incidence of malformations and a high death rate during the perinatal period. To investigate the underlying mechanisms for abnormal development of cloned pig fetuses, we compared body weight, amniotic fluid (AF) metabolome, and placental transcriptome between SCNT- and artificial insemination (AI)-derived pig fetuses. Results showed that the body weight of SCNT pig fetuses was significantly lower than that of AI pig fetuses. The identified differential metabolites between the two groups of AF were mainly involved in bile acids and steroid hormones. The levels of all detected bile acids in SCNT AF were significantly higher than those in AI AF. The increase in the AF bile acid levels in SCNT fetuses was linked with the downregulation of placental bile acid transporter expression and the abnormal development of placental folds (PFs), both of which negatively affected the transfer of bile acids from AF across the placenta into the mother's circulation. Alteration in the AF steroid hormone levels in cloned fetuses was associated with decreased expression of enzymes responsible for steroid hormone biosynthesis in the placenta. In conclusion, cloned pig fetuses undergo abnormal intrauterine development associated with alteration of bile acid and steroid hormone levels in AF, which may be due to the poor development of PFs and the erroneous expression of bile acid transporters and enzymes responsible for steroid hormone biosynthesis in the placentas.

Cloned pig fetuses exhibit fatty acid deficiency from impaired placental transport.

Cloned pig fetuses produced by somatic cell nuclear transfer show a high incidence of erroneous development in the uteri of surrogate mothers. The mechanisms underlying the abnormal intrauterine development of cloned pig fetuses are poorly understood. This study aimed to explore the potential causes of the aberrant development of cloned pig fetuses. The levels of numerous fatty acids in allantoic fluid and muscle tissue were lower in cloned pig fetuses than in artificial insemination-generated pig fetuses, thereby suggesting that cloned pig fetuses underwent fatty acid deficiency. Cloned pig fetuses also displayed trophoblast hypoplasia and a reduced expression of placental fatty acid transport protein 4 (FATP4), which is the predominant FATP family member expressed in porcine placentas. This result suggested that the placental fatty acid transport functions were impaired in cloned pig fetuses, possibly causing fatty acid deficiency in cloned pig fetuses. The present study provides useful information in elucidating the mechanisms underlying the abnormal development of cloned pig fetuses.

DZNep and UNC0642 enhance in vitro developmental competence of cloned pig embryos.

Somatic cell nuclear transfer in mammalian cloning suffers from a faulty epigenetic reprogramming, which is believed to cause developmental failures in cloned embryos. Regulating the epigenetic-modifying enzymes can rescue the chromatin of cloned embryos from aberrant epigenetic status, thereby potentially promoting cloning efficiency. In this study, we investigated the effect of two histone methyltransferase inhibitors, namely, DZNep and UNC0642, on the in vitro developmental competence of cloned pig embryos. We found that (1) treatment with 10 nM DZNep or 5 nM UNC0642 for 24 h after activation had the best promoting effect on the development of cloned embryos (blastocyst rate 10.32% vs 18.08% for DZNep, and 10.44% vs 18.14% for UNC0642); (2) 10 nM DZNep and 5 nM UNC0642 significantly decreased the levels of H3K27me3 and H3K9me2, respectively, at the 2-cell, 4-cell and blastocyst stages; (3) the apoptosis level was lower in the treatment groups than in untreated control; and (4) the transcriptional expression of epigenetic genes (EZH2, GLP, G9a, Setdb1, Setdb2, Suv39h1 and Suv39h2) was decreased and pluripotency genes (Nanog, Pou5f1, Sox2 and Bmp4) was increased in treatment groups compared with control. These results indicated that treatment with DZNep and UNC0642 improves the epigenetic reprogramming of cloned embryos, which could render beneficial effect on the embryo quality and aberrant gene expression, and finally improve the developmental competence of cloned pig embryos.

Characterization and comparative analyses of transcriptomes of cloned and in vivo fertilized porcine pre-implantation embryos.

Somatic cell nuclear transfer (SCNT) is the only method known to rapidly reprogram differentiated cells into totipotent embryos. Most cloned embryos become arrested before implantation and the details of the underlying molecular mechanism remain largely unknown. Dynamic regulation of the transcriptome is a key molecular mechanism driving early embryonic development. Here, we report comprehensive transcriptomic analysis of cloned embryos (from Laiwu and Duroc pigs) and in vivo fertilized embryos (from Duroc pigs) using RNA-sequencing. Comparisons between gene expression patterns were performed according to differentially expressed genes, specific-expressed genes, first-expressed genes, pluripotency genes and pathway enrichment analysis. In addition, we closely analyzed the improperly expressed histone lysine methyltransferases and histone lysine demethylases during cell reprogramming in cloned embryos. In summary, we identified altered gene expression profiles in porcine cloned pre-implantation embryos in comparison to normal in vivo embryos. Our findings provide a substantial framework for further discovery of the epigenetic reprogramming mechanisms in porcine SCNT embryos.

Maternal dietary supplementation of arginine increases the ratio of total cloned piglets born to total transferred cloned embryos by improving the pregnancy rate of recipient sows.

The extremely low full-term developmental efficiency of cloned pig embryos limits the practical application of pig cloning techniques. Maternal dietary supplementation of the nutritionally important amino acid, arginine, can enhance prenatal developmental rate of in vivo fertilization-derived pig embryos. It was hypothesized that maternal dietary addition of arginine can also improve the developmental capacity of cloned pig embryos. To test this hypothesis, there was a comparison of the reproductive performance between recipient sows fed an L-arginine-supplemented diet (L-Arg group) and those fed the control diet (control group). There was a subsequent comparison of the developmental indexes of cloned piglets farrowed in the L-Arg and control groups of surrogate sows. Dietary supplementation of L-arginine during gestation days 14-75 increased the plasma concentrations of arginine and arginine metabolites, including nitric oxide, spermidine, and putrescine in recipient sows of transferred cloned pig embryos. Although maternal arginine addition did not affect the birth weight and placental development indexes of newborn cloned piglets, it significantly increased the ratio of total cloned piglets born to total transferred cloned pig embryos by increasing the pregnancy rate of recipient sows. The results of this study suggest that nutritional management of recipient sows is an effective approach to improve the developmental rate of cloned pig embryos.

Birth weight, umbilical and placental traits in relation to neonatal loss in cloned pigs.

Cloned piglets generated through somatic cell nuclear transfer (SCNT) have a high rate of neonatal death. Postnatal loss is associated with low birth weight, umbilical status and placental parameters in fertilisation-derived piglets. To investigate whether or not this relationship also exists in cloned piglets, birth weight, umbilical status, placental parameters, placental morphology and gene expression pattern were compared among four piglet groups, namely, SCNT-derived male piglets that died within 4 days (SCNT-DW4), SCNT-derived male piglets that survived over 4 days (SCNT-SO4), artificial insemination (AI)-generated male piglets that died within 4 days (AI-DW4) and AI-generated male piglets that survived over 4 days (AI-SO4). Results showed that the occurring frequency of abnormal umbilical cord in SCNT-DW4 piglets was significantly higher than that in AI-SO4 piglets but was similar to that in SCNT-SO4 and AI-DW4 piglets. The birth weight, placental surface area and placental weight of AI-SO4, AI-DW4 and SCNT-SO4 groups were similar but were significantly higher than those in SCNT-DW4 group. SCNT-SO4 placentas exhibited mild but SCNT-DW4 placentas showed severe morphological abnormalities compared with AI-SO4 placentas. The expression profiles of imprinting, angiopoiesis, nutrient transport, apoptosis and oxidative stress-related genes in SCNT-DW4 placentas were erroneous compared with those in SCNT-SO4 and AI-SO4 placentas, which both had similar gene expression patterns. These results indicate that birth weight, umbilical status, placental parameters, placental morphology and gene expression were associated with neonatal death of cloned piglets. The high loss of cloned piglets during neonatal age may be caused by severe deficiency of extra-embryonic development during prenatal stage.