[Research progress on infiltrating zone and microvascular invasion of hepatic alveolar echinococcosis].

Hepatic alveolar echinococcosis (AE) is a parasitic disease with biological characteristics similar to malignant tumor. It has no obvious clinical symptoms in the early stage. Most patients have complications such as jaundice, ascites and gastrointestinal bleeding when they see a doctor. At this time, the course of disease is at an advanced stage. In addition, the incomplete resection of the AE lesion(s) leads to a high postoperative recurrence rate, which has a serious impact on the physical and mental health of patients. Based on the summary of the latest research at home and abroad and the analysis of blood supply, microvascular invasion and vascular growth factor expression in the "infiltrating zone" adjacent to the lesions of hepatic AE, this article has a deep understanding of the occurrence and development process of hepatic AE, aiming to better guide clinical practice and improve the quality of life of patients.

Effects of the histone methyltransferase inhibitor UNC0638 on histone H3K9 dimethylation of cultured ovine somatic cells and development of resulting early cloned embryos.

Aberrant hypermethylation of histone H3 lysine 9 (H3K9) may be involved in the developmental failure of cloned embryos. UNC0638 is a type of small molecule that can specifically inhibit the enzyme activity of histone methyltransferase EHMT2 and reduce the H3K9 dimethylation (H3K9me2) levels in cells. The objective of this study was to investigate the effect of UNC0638 in regulating H3K9me2 and development of cloned embryos. Results showed that UNC0638 could efficiently reduce H3K9me2 levels of cultured sheep foetal fibroblast cells in a concentration-dependent manner. Cloned embryos were subsequently produced from UNC0638-treated donor cells with down-regulated H3K9me2, but their in vitro development was not improved when compared with the control. Our study suggested that revision of the single histone H3K9me2 modification may be not sufficient for rescuing the development of cloned embryos. However, because of its low cellular toxicity, UNC0638 may still be a potential chemical that could be used in regulating epigenetic modification of cloned embryos.

Abnormal histone H3K9 dimethylation but normal dimethyltransferase EHMT2 expression in cloned sheep embryos.

The objective was to investigate the relationship between histone H3 lysine 9 (H3K9) dimethylation (me2) and the histone methyltransferase EHMT2 (also known as G9A) in ovine embryos cloned by somatic cell nuclear transfer (SCNT). Levels of H3K9me2 or EHMT2 were detected (with immunostaining) and compared between SCNT and IVF-derived preimplantation embryos. In one-cell embryos, SCNT zygotes had significantly higher levels of H3K9me2 and EHMT2 than IVF zygotes. In cloned embryos, H3K9me2 remained hypermethylated relative to IVF embryos at two-cell and late developmental stages (morula and blastocyst), with no difference (P > 0.05) between IVF and SCNT embryos in EHMT2 levels from two-cell to blastocyst stages. The EHMT2-specific inhibitor, BIX01294, reduced global H3K9me2 levels in cultured ovine cells or SCNT embryos, but it was not appropriate for somatic cell nuclear transfer because of its high cellular toxicity. We inferred that abnormal H3K9me2 hypermethylation in SCNT embryos may not completely arise from EHMT2 expression error.

Association of distributions of three types of germinal vesicle stage oocytes with the canine follicle location in the ovary.

The objective of current study was to compare the nuclear configurations of canine oocytes recovered from between follicles after isolation. Follicles isolated were classified into follicle-S (follicles located in the ovarian surface) and follicle-I (follicles located inside the ovary) based on the follicle location in the ovary. Nuclear stages of canine oocytes recovered from follicle-S and follicle-I were examined by phase-contrast microscopy after isolation. Results demonstrated that canine GV stage oocytes can be classified into three types based on the status of the nuclear envelope, nucleolus, and chromatin: type A, type B, and type C. In follicle-S group, the majority (95.5%) of canine GV stage oocytes was of type B. All canine GV stage oocytes recovered from follicle-S (including type B and type C) were characterized by nuclear envelope disappearance prior to nucleolus collapse. In contrast, in follicle-I group, the majority (60.2%) of canine GV stage oocytes was of type C. Unexpectedly, a small proportion of canine GV stage oocytes from follicle-I (donated type A) were characterized by nuclear envelope disappearance following nucleolus collapse. In conclusion, nuclear configurations of each type of canine GV stage oocytes may differ from each other. Distributions of each type of canine GV stage oocytes may associate with the follicle location in the ovary.

Full-term development of rabbit embryos produced by ICSI with sperm frozen in liquid nitrogen without cryoprotectants.

The aim of the present study was to establish the technology of intracytoplasmic sperm injection (ICSI) in rabbit by using the sperm frozen without cryoprotectants. Observation under an electron microscope revealed that the rabbit spermatozoa frozen without cryoprotectants had severe damage especially in the plasma membrane and junction between head and tail. However, after being injected into the oocytes, the sperm frozen without cryoprotectants retained the capability of supporting the cleavage and development of the ICSI oocytes, with no significant difference from that of fresh sperm, although the development of ICSI embryos derived from either frozen sperm or fresh sperm is much lower than that of in vivo-fertilized zygotes. When additional artificial activation was applied following ICSI, the rates of cleavage and blastocyst formation of ICSI oocytes were significantly increased when compared with the oocytes without additional activation. Yet, the cell numbers in blastocysts were not significantly different between the activation and non-activation group. After embryo transfer, four offspring were obtained from the oocytes microinjected with the sperm frozen without cryoprotectants. The technology established by this study may facilitate exploring the ICSI-based transgenic method in rabbit and broaden the application of ICSI technique in related field.

Technical note: transfer of ovine embryos through a simplified mini-laparotomy technique.

The aim of this experiment was to examine whether a simplified mini-laparotomy technique is suitable for embryo transfer in ewes. In vitro produced blastocysts were transferred to the uterine horns of synchronous recipient ewes. Each recipient received 1 embryo by conventional laparotomy (n = 36), laparoscopy (n = 21), or by simplified mini-laparotomy (n = 33). Pregnancy rates for these 3 transfer techniques were 38.9, 47.6, 45.5%, respectively (P = 0.58). Of these techniques, the simplified mini-laparotomy was preferred because of benefits it provided in terms of savings in time and expense as well as reductions in surgical trauma and elimination of exteriorization of most of the reproductive system along with only one suture. It is likely that this technique will play a substantial role in adoption of embryo transfer by the sheep industry.

Electroejaculation and semen characteristics of Asiatic black bears (Ursus thibetanus).

This study details the seminal traits in the rare Asiatic black bear, revealing that this species generally produces high quality, concentrated ejaculates containing sperm of high motility and morphological integrity and similar to other members of the Ursine lineage. Semen was collected by electroejaculation and 23 trials were performed on 18 bears. The mean values were obtained for volume (0.61 ml), pH (7.1), sperm concentration (1049 x 10(6) ml(-1)), total sperm counts (502.8 x 10(6)), motility percentage (63.8%), forward progressive status (3.5), abnormal sperm percentage (37.9%) and intact acrosome percentage (76.6%).

DNA methylation patterns in in vitro-fertilised goat zygotes.

Recent studies have shown that zygote demethylation patterns in mammals are variable among species. However, the methylation patterns of goat zygotes have not yet been reported on. In the present study, using immunofluorescence staining with an antibody against 5-methylcytosine, the methylation patterns of in vitro-derived goat zygotes were studied. The results indicate that goat zygotes do not undergo active global demethylation during pronuclei development, which is similar to the situation in ovine, but not in murine or bovine zygotes. This is believed to be the first report regarding methylation in goat zygotes.

Transgenic twin lambs cloned by granulosa cells.

The development potential of transgenic adult cells after nuclear transfer (NT) was evaluated. Primary ovine granulosa cells (GC(S)) from a slaughter ovary were transfected with pEGFP-N1 plasmid DNA. Three G418-resistance cell lines (A2, B2 and B4) were used as donor cells in NT. A total of 162 NT blastocysts were then frozen with ethylene glycol solution and stored for five months before transplanted into recipients. Twenty-nine frozen thawed NT blastocysts were transferred into 15 synchronized recipients. Twin lambs (6.9%) derived from B2 line were delivered by cesarean section on day 143 but died after birth. A tumor consisting of lung tissues was found on the surface of left lung of the 4-kg lamb and histological analysis indicated that it resembles a hamartoma. DNA analysis confirmed that two lambs were genetically identical to B2 donor cells. Gene insertion and expression have been detected in fibroblasts cells derived from muscle tissues of the lambs. This study indicates that granulosa cell is a suitable cell type for producing transgenic animals by nuclear transfer. Offspring were produced after long-term storage of NT blastocysts.

Extraction of sp18 family membrane proteins on posterior head of bull sperm and the effect of anti-sp18 IgG on sperm motility and murine in vitro fertilization.

Bovine sperm heads were separated via ultrasonic treatment and centrifugation. Anti-bull sperm IgG was produced by immunizing rabbits with acrosome-reacted bull sperm heads. SDS PAGE patterns revealed that the main membrane proteins on acrosome-reacted bull sperm head were sp18 family, including 18, 16, and 14 kD, which represented about 64% of the total membrane proteins in bull sperm. Indirect immunofluorescence shown sp18 antigens primarily distributed in postacrosomal and proximal tail regions. Western blot analysis revealed that the anti-bull sperm IgG reacted with sp18 antigens in acrosome-reacted bull sperm head and bull seminal plasma. Anti-bull sperm IgG also reacted with 14, 16, 18, 42, 57 and 60 kD proteins in fresh bull, mouse and rabbit sperm. Anti-sp18 IgG caused agglutination of bull and rabbit sperm, but had no effect on murine sperm. In murine in vitro fertilization trials, preincubating capacitated sperm with 0.364 mg/ml of anti-sp18 IgG resulted in a decrease in the fertilization rate from 75.6% in the controls to 50.8% in the experimental groups (p < 0.001).

Mice lacking osteopontin show normal development and bone structure but display altered osteoclast formation in vitro.

We have used homologous recombination in embryonic stem cells to generate mice with a targeted disruption of the osteopontin (Opn, or Spp1, for secreted phosphoprotein 1) gene. Mice homozygous for this disruption fail to express osteopontin (OPN) as assessed at both the mRNA and protein level, although an N-terminal fragment of OPN is detectable at extremely low levels in the bones of -/- animals. The Opn -/- mice are fertile, their litter size is normal, and they develop normally. The bones and teeth of animals not expressing OPN are morphologically normal at the level of light and electron microscopy, and the skeletal structure of young animals is normal as assessed by radiography. Ultrastructurally, proteinaceous structures normally rich in OPN, such as cement lines, persist in the bones of the Opn-/- animals. Osteoclastogenesis was assessed in vitro in cocultures with a feeder layer of calvarial osteoblast cells from wild-type mice. Spleen cells from Opn-/- mice cells formed osteoclasts 3- to 13-fold more frequently than did control Opn+/+ cells, while the extent of osteoclast development from Opn -/- bone marrow cells was about 2- to 4-fold more than from the corresponding wild-type cells. Osteoclast development occurred when Opn-/- spleen cells were differentiated in the presence of Opn-/-osteoblasts, indicating that endogenous OPN is not required for this process. These results suggest that OPN is not essential for normal mouse development and osteogenesis, but can modulate osteoclast differentiation.

Osteopontin-induced modifications of cellular functions.

Osteopontin (OPN) serves both a cell attachment function and a cell signalling function via the alpha v beta 3 integrin. We have investigated the action on mammalian cells of recombinant OPN made both in E. coli and in human cells. In its cell signalling capacity it initiates a signal transduction cascade that includes changes in the intracellular calcium ion levels and the tyrosine phosphorylation status of several proteins including pp60src and components of focal adhesion complexes. Effects on gene expression include suppression of the induction of nitric oxide synthase by inflammatory mediators. OPN can also reduce cell peroxide levels, promote the survival of cells exposed to hypoxia, and inhibit the killing of tumor cells by activated macrophages.