The dynamic pattern of PLIN3 in pig oocytes and cumulus cells during in vitro maturation.

Lipid droplets (LDs) are the main energy resource for porcine preimplantation embryonic development. PLIN3 has been implicated in LD formation and regulation. Therefore, this study aimed to detect the dynamic pattern of PLIN3 in pig oocytes and cumulus cells (CC) during in vitro maturation (IVM), and to determine the relationship between PLIN3 and LD content. IVM with cumulus-enclosed oocytes (CEO), cumulus-denuded oocytes (DO) and the CCs denuded from the corresponding oocytes (DCC) was performed in porcine follicular fluid (PFF) or PFF-free optimized medium. DO and the DCC were cultured together under the same conditions as described above, while the DO was named DTO and the DCC was named DTCC in this group. Firstly, our results revealed LDs distributed widely in oocytes and CC, while the PLIN3 protein coated these LDs and spread out ubiquitously in the cytoplasm. Secondly, not only the mRNA level but also at protein level of PLIN3 in immature naked oocytes (IO) was higher than that in matured CEO, DO and DTO. Although PLIN3 was expressed at lower levels in CC from immature oocytes (ICC), the protein level of PLIN3 was comparably higher in the ECC and DCC groups. The triglyceride (TG) content in CEO and DO was significantly less abundant compared with that in IO. Therefore, our results indicated that co-culturing of oocytes and CC might affect PLIN3 expression levels in CC but not in oocytes. Lipid accumulation in pig oocytes during maturation might be affected by PLIN3 cross-talk between oocytes and CC.

Symptomatic lumbar mobile segment with spinal canal stenosis in a fused spine associated with diffused idiopathic skeletal hyperostosis.

BACKGROUND CONTEXT: Chronic, continuous stress at the junction of a stable/unstable site of the spine in diffuse idiopathic skeletal hyperostosis (DISH) has been reported to cause a nonunion. Back pain resulting from the nonunion has been rarely reported and few operative treatments have been suggested. PURPOSE: To report and discuss the pathogenesis, treatment, and surgical outcome of a rare cause of back pain. STUDY DESIGN: Case report of back pain caused by a single lumbar segment is lacking bony union at the caudal end of a fused spine associated with diffuse idiopathic skeletal hyperostosis. METHODS: Back pain in a 66-year-old man who had suffered for 10 years worsened. The back pain and thigh pain became intolerable, and the left buttock and thigh became numb. Radiographs and computed tomography images showed continuous hyperostosis in the anterior aspect of the vertebral bodies from C2 to L2. At the caudal adjacent level of these fused segments, L2/3 level was mobile and had canal stenosis. Decompression and posterior lumbar interbody fusion (PLIF) were performed. RESULTS: The pain disappeared soon after the operation. The nonunited segment showed bony union at the 5-year follow-up. CONCLUSIONS: PLIF may be an option for surgically treating symptomatic nonunited lumbar segments at the caudal end of a fused spine with DISH in cases unresponsive to conservative treatment.

Genome-scale identification of nucleosome organization by using 1000 porcine oocytes at different developmental stages.

The nucleosome is the basic structural unit of chromosomes, and its occupancy and distribution in promoters are crucial for the regulation of gene expression. During the growth process of porcine oocytes, the "growing" oocytes (SF) have a much higher transcriptional activity than the "fully grown" oocytes (BF). However, the chromosome status of the two kinds of oocytes remains poorly understood. In this study, we profiled the nucleosome distributions of SF and BF with as few as 1000 oocytes. By comparing the altered regions, we found that SF tended toward nucleosome loss and more open chromosome architecture than BF did. BF had decreased nucleosome occupancy in the coding region and increased nucleosome occupancy in the promoter compared to SF. The nucleosome occupancy of SF was higher than that of BF in the GC-poor regions, but lower than that of BF in the GC-rich regions. The nucleosome distribution around the transcriptional start site (TSS) of all the genes of the two samples was basically the same, but the nucleosome occupancy around the TSS of SF was lower than that of BF. GO functional annotation of genes with different nucleosome occupancy in promoter showed the genes were mainly involved in cell, cellular process, and metabolic process biological process. The results of this study revealed the dynamic reorganization of porcine oocytes in different developmental stages and the critical role of nucleosome arrangement during the oocyte growth process.

LC3-Dependent Autophagy in Pig 2-Cell Cloned Embryos Could Influence the Degradation of Maternal mRNA and the Regulation of Epigenetic Modification.

In this study, the distribution as well as the effect of autophagy on reprogramming in pig cloned embryos were observed immediately after somatic cell nuclear transfer. Results showed that the LC3 was at the highest level in cloned embryos at 2-cell stage, and it decreased with the development from 2-cell stage to blastocyst. Different to cloned embryos, the intensity of LC3 in parthenogenetic activation (PA) embryos was at the highest level at 4-cell stage. A markedly higher level of Bmp15, H1foo, and Dppa3 was shown in cloned embryos at 2-cell stage (p < 0.05 or p < 0.01), but a significantly lower level of LC3, Sox2, and eIF1A was observed at 4-cell stage (p < 0.05), compared with PA embryos. When the efficient interfering by the LC3 siRNA was performed on the cloned embryos (p < 0.01), not only the mRNA level of maternal Cyclin B, Bmp15, Gdf9, c-mos, H1foo, and Dppa3 was increased significantly (p < 0.05), but also the expression of Dnmt1 and Dnmt3b was obviously upregulated (p < 0.05). Although the expression of Sox2 and Oct4 is not changed, the expression of Stat3 decreased significantly (p < 0.05). Furthermore with the treatment of 200 nM rapamycin, the expression of eIF1A and Stat3 was significantly increased at 4-cell stage. In conclusion, the LC3-dependent autophagy mainly occurred in cloned embryos at 2-cell stage, but at 4-cell stage in PA embryos. In addition, the modulation of autophagy could affect genome activation by influencing the degradation of maternal mRNA and regulating the expression of DNA methyltransferase.

Dynamic Reorganization of Nucleosome Positioning in Somatic Cells after Transfer into Porcine Enucleated Oocytes.

The nucleosome, the fundamental structural unit of chromatin, is a critical regulator of gene expression. The mechanisms governing changes to nucleosome occupancy and positioning during somatic cell reprogramming remain poorly understood. We established a method for generating genome-wide nucleosome maps of porcine embryonic fibroblasts (PEF), reconstructed 1-cell embryos generated by somatic cell nuclear transfer (SCNT), and fertilized zygotes (FZ) using MNase sequencing with only 1,000 cells. We found that donor PEF chromatin, especially X chromosome, became more open after transfer into porcine oocytes and nucleosome occupancy decreased in promoters but increased in the genic regions. Nucleosome arrangements around transcriptional start sites of genes with different expression levels in somatic cells tended to become transcriptionally silent in SCNT; however, some pluripotency genes adopted transcriptionally active nucleosome arrangements. FZ and SCNT had similar characteristics, unlike PEF. This study reveals the dynamics and importance of nucleosome positioning and chromatin organization early after reprogramming.

Distribution and content of lipid droplets and mitochondria in pig parthenogenetically activated embryos after delipation.

The present study examines the effect of delipation on developmental competence and the distribution pattern of lipid droplets (LDs) and mitochondria in parthenogenetically activated (PA) pig embryos. Mature oocytes were delipated by centrifugation after partial digestion of the zonae pellucidae, subjected to parthenogenetic activation after total removal of zonae pellucidae by pronase, and then cultured in vitro up to the blastocyst stage. The contents and distributions of LDs and mitochondria in the oocytes and/or embryos were observed by staining with Oil Red O and MitoTracker Red CMXRos, respectively. The LD and mitochondrial contents were significantly reduced by the delipation process, and only smaller LDs remained in the delipated oocytes and/or embryos. Their content remained constant from the metaphase II oocyte to the blastocyst stage, but they became gradually smaller as the oocytes and/or embryos developed. The distribution pattern of the LDs in the delipated embryos changed over time and in a manner different to that seen in the controls. In the early developmental stages (1- to 4-cell stages), they were distributed peripherally and formed a ring around the nucleus. However, by the blastocyst stage, a homogeneous distribution of LDs was observed in both the inner cell mass and trophectoderm. The distribution pattern of mitochondria also changed with the development of the delipated PA embryos and again, in ways different to those seen in the controls. In the early 1- to 4-cell stages, a peripheral distribution of mitochondrial foci was observed in each blastomere. However, in blastocysts, the mitochondria were homogeneously distributed throughout the inner cell mass and trophectoderm. Although the cleavage rate at the 2- and 4-cell stages of the PA embryos was not affected by delipation (95.83 ± 2.25% vs. 97.44 ± 0.67%; 79.17 ± 4.47% vs. 84.62 ± 1.19%), it was reduced significantly in the blastocyst compared with the controls (21.67 ± 3.78% vs. 49.36 ± 1.77%). The distribution pattern of the LDs in oocytes and/or embryos at different developmental stages, and that of the mitochondria in metaphase II oocytes, was affected by delipation. The developmental competence of porcine PA embryos would appear to be affected by delipation.

[The application of reconstruction with autograft implantation in total hip replacement with regional acetabular deficiency].

OBJECTIVE: To evaluate the effect of reconstruction with autograft implantation in total hip arthroplasty(THA) with regional acetabular deficiency. METHODS: From 1991 to 2000, 39 cases of THA with acetabular deficiency were conducted. Autogenous bone implantation was used to reconstruct the deficient acetabulum. Of the 39 patients, 25 were males and 14 were females. The age ranged from 34 to 62 (45.2 on average). There were 21 cases of developmental dysplasia resulted deficiency, 14 cases of fracture of femoral neck complicated with head necrosis (10 hips) and fracture of acetabulum(4 hips). The resected femoral heads or autologous ilium were made the wedge-shaped graft and implanted into the deficient acetabulum, which included 12 cases with cement THA and 27 with cementless THA. Of all the cases, 24 were followed up 2 to 10 years (6.7 years on average). Harris scores before operation were 18 to 50 (38.1 on average). RESULTS: The limbs were lengthened by 2.4 cm on average. No serious complications were observed in these patients. Compared with the scores before the operation, the average Harris scores after the operation were 92.1 (P < 0.01)and 86.3 (P < 0.05) in the one-year and the latest follow-up respectively. The rates for the good were 91.7% and 83.3% in the one-year and the latest follow-up respectively. CONCLUSION: The acetabular reconstruction with autograft in THA will bring better stability in those patients with acetabular deficiency.