Porcine androgenetic embryos develop to fetal stage in recipient mothers.

In livestock, parthenogenic embryos are simple to produce, but androgenetic embryos have been successfully produced only in sheep and cows. In the present study, matured porcine oocytes were enucleated by micromanipulation and then fertilized with sperm in vitro, thereby producing porcine androgenetic embryos. Porcine androgenetic embryos, which had only sperm genomes, were assessed for cleavage and for blastocyst formation 2 and 6 d after IVF, respectively. There was no difference in cleavage rate between androgenetic embryos and biparental IVF embryos (mean ± SD androgenetic: 65.5 ± 5.4%; biparental IVF: 63.2 ± 3.6%), but there was a difference in the rate of blastocyst formation (androgenetic: 4.5 ± 0.7%; biparental IVF: 30.2 ± 2.6%, P < 0.05). The average number of cells in Day 6 androgenetic blastocysts (34.3 ± 18.2) was lower (P < 0.05) than that in biparental IVF blastocysts (44.1 ± 19.5), but did not differ from that in parthenogenetic embryos (35.7 ± 16.7). The androgenetic embryos were transferred into recipient mothers to examine the competence of post-implantation development. Androgenetic fetuses were present on Days 21 and 25, but not on Days 28, 31, or 35. Of the six androgenetic fetuses recovered on Day 21, five had normal, translucent bodies, and two of these five had beating hearts. The four fetuses recovered on Day 25 were all non-viable. In conclusion, porcine androgenetic embryos initiated embryogenesis and had reached a viable fetal stage 21 days after IVF.

A simple method for producing tetraploid porcine parthenogenetic embryos.

The objective was to produce porcine tetraploid parthenogenetic embryos using cytochalasin B, which inhibits polar body extrusion. Porcine cumulus-enclosed oocytes aspirated from antral follicles were cultured for 51 h, and treated with cytochalasin B from 35 h to 42 h after the start of culture. After maturation culture, 74.7% (2074/2775) of oocytes treated with cytochalasin B did not extrude a polar body (0PB oocytes). In contrast, 80.4% (1931/2403) of control oocytes extruded a polar body (1PB oocytes). The 0PB oocytes were electrically stimulated, treated with cytochalasin B again for 3 h, and then cultured without cytochalasin B. Six days after electrical stimulation, 49.8% (321/644) reached the blastocyst stage. The number of cells in these blastocysts derived from 0PB oocytes was significantly lower than that from 1PB oocytes (0PB: 24.9 ± 10.6; 1PB: 43.0 ± 17.1; mean ± SD). A porcine chromosome 1-specific sequence was detected in parthenogenetic 0PB embryos by fluorescence in situ hybridization (FISH) analysis. Typical pronucleus-stage samples derived from 0PB embryos had two pronuclei, each with two signals. In two-cell and blastocyst-stage embryos, four signals were detected in each nucleus derived from 0PB embryos. We inferred that 0PB oocytes, which had a tetraploid number of chromosomes, started to develop as tetraploid parthenotes after electrical stimulation, and that tetraploid status was stably maintained during early embryonic development, at least until the blastocyst stage.

Identification of genes downregulated during differentiation of porcine mesenteric adipocytes.

Adipose tissue development is a process that comprises not only hypertrophy, but also hyperplasia, of adipocytes. Although the proliferation of undifferentiated preadipocytes plays an important part in hyperplasia, this process is less well understood than the post-proliferation differentiation process. Despite the potential importance of porcine visceral adipose tissue to both meat production and biomedical research, there has been little study of this tissue and, in particular, its development and differentiation. To detect the genes involved in the maintenance of porcine visceral preadipocytes in an undifferentiated state or in the inhibition of adipocyte differentiation, we performed suppression subtractive hybridization using mesenteric preadipocytes in which fragments of the genes that are downregulated at 2 d of differentiation were enriched. We selected 672 clones and subjected them to differential screening and semiquantitative reverse transcription (RT)-PCR. As a result, we identified 34 downregulated genes. Among these, the detailed expression patterns of 6 genes were examined using real-time RT-PCR in both preadipocytes during in vitro differentiation and cell fractions directly isolated from pig mesenteric adipose tissue. The expressions of connective tissue growth factor, AXL receptor tyrosine kinase, stromal membrane-associated protein 1-like, and retinoic acid-induced 14 were significantly downregulated during adipocyte differentiation in vitro (P < 0.05), and the expressions of Rho/Rac guanine nucleotide exchange factor 2 and secreted frizzled-related protein 4 also tended to be decreased, although not significantly. Furthermore, all 6 genes showed significantly greater expression in stromal vascular cells, which contain preadipocytes, than in mature adipocytes (P < 0.05), raising the possibility that these genes are involved in adipocyte differentiation in vivo as well as in vitro.