[Interpretation and analysis of traditional Chinese medicine injection instructions].

Traditional Chinese medicine injection(TCMI) is a new preparation developing recent years, which is spreading because of the significant effect. Doctors acquire information of medicine from the prospectuses mainly. But due to many reasons, there are a lot of shortcomings such as lack of items, unknown of composition, the narrow scope, etc. Unreasonable drug use in field and dose happened frequently. Adverse reactions are often reported. They restricted the use of TCMI severely. We must pay attention to the market evaluation of TCMI, and improve the prospectuses gradually on the basis of clinical evidence. One hand, the use of drugs can be reference to it. On the other hand, it can avoid unreasonable use of drugs. We have done it in order to contribute to the clinical application of TCMI.

[Effect of intact donor cell membrane and cytoplasm of cumulus on development of mouse somatic clones in vitro].

Reconstructed embryos can be got by two nuclear transfer methods, fusion and microinjection. However, the fusion method is time-taking and has low efficiency. Microinjection involved the donor nucleus isolation, which may lead to the damage of the nucleus DNA. Cloning efficiency may be reduced by low fusion rate of the cell fusion method and damaged DNA of microinjection method. So some researchers used the intracytoplasmic injection of the whole-cell donor, which was simple and less labor-intensive for cloning study. Here we used the intact cumulus cell as donor to study the feasibility of the microinjection method in mouse nuclear transfer. After the whole cell injection of the cumulus, most of the couplets were fragmented following activation at 1 or 6 h after injection. Whereas those only the nucleus after isolation from the cumulus was injected cleaved and developed normally. The control group of oocytes, which were not injected with the donor, was also fragmented after enucleation. Most of the couplets of the parthenotes after intact cumulus cell injection were fragmented. The group of those in which the donor was just broken and then injected (nucleus with most of the broken membrane and the cytoplasm were injected) developed as a lower rate compared to those only isolated nucleus being injected. These results suggest that the intact donor membrane of cumulus cell hinder the reprogramming of oocyte for somatic nucleus and components of donor cytoplasm and membrane affected the development of the clones in vitro.

[Maternal factors affecting development of early mouse embryos].

In vertebrates, oocytes undergo a series of maturation steps, arresting at metaphase II, and can then be fertilized by a sperm. Fertilization initiates molecular events that lead to the activation of early embryonic development. Fertilized oocytes or activated reconstituted embryos then activate the zygotic genome, a crucial event that initiates early embryonic development. The functions of the maternal factors derived from oocytes are different at various mouse embryonic developmental stages. Mouse zygotic genome is activated at the two-cell stage which implies that embryonic development is transferred from the oocyte itself to the embryo. Sometimes mouse embryos are blocked at the two-cell stage, for which the mechanism is not clear. So exploring the functions of some maternal factors in the two-cell stage embryos may help us to understand the potential reasons for early embryonic development failure. Reprogramming a foreign and terminally differentiated somatic nucleus by transferring it to the enucleated oocyte cytoplasm triggers epigenetic changes that eventually lead to the birth of a viable animal. This indicates the oocyte cytoplasm plays a critical role in the development of reconstructed embryos.

[The effects of genomic imprinting on animal development and cloning].

In placental mammals, there is a small group of special genes, which are imprinted so that only one of the parental alleles is actually expressed in target cells. This epigenetic process is named genomic imprinting. Till now, about eighty genes are found to be imprinted in mice and human, and imprinting is conserved in ruminant species as well. This paper emphasized the effects of genomic imprinting on development and animal cloning, exhibited the function of imprint genes by analyzing their origin, and discussed their formation mechanism for understanding the profound effects of this epigenetic modification on development. Many imprinted genes are involved in fetal development, and may influence fetal growth and behavior. Imprinting appears to be particularly important for placental development. Epigenetic deregulation of imprinting may lead to complex diseases in human. And there is now a large body of evidence for loss of appropriate imprinting in numerous tumors. So far, nuclear transfer is a very inefficient process. The rate of cloned animals' surviving was very poor, and there were also frequent inherent anomalies. The most common abnormal phenotypes had the similar characteristics of the consequence due to deregulated expression of imprinted genes,indicating that the key factor to limite cloning efficiency might be the aberrant expression of imprinted genes.

Reduced polyspermic penetration in porcine oocytes inseminated in a new in vitro fertilization (IVF) system: straw IVF.

High incidence of polyspermy is still a major problem in the in vitro fertilization (IVF) of porcine oocytes matured in vitro. This study was designed to examine whether embryo cryopreservation straws can be used to conduct IVF in porcine oocytes. The efficiency of this system was further compared with traditional microdrop IVF. Immature oocytes were aspirated from antral follicles and matured in vitro. After maturation, oocytes were inseminated either in straws or in microdrops with frozen-thawed boar spermatozoa. For straw IVF, sperm concentration and the presence of air columns between insemination segment and oil column were examined. Sperm-oocyte binding and cortical granules (CGs) before and after sperm penetration were examined by confocal microscopy. When various sperm concentrations were used for IVF in the straws with air columns, it was found that 5 x 106 cells/ml of sperm concentration was the optimal concentration; a high penetration rate (94.0%) and normal fertilization (oocytes with both male and female pronuclei) rate (38.2%) were obtained. Increasing sperm concentration to 10 x 106 cells/ml increased polyspermic penetration (61.9%) without affecting sperm penetration (86.9%). Reducing sperm concentration to 1 x 106 cells/ml reduced polyspermic penetration (25.6%), but sperm penetration rate (69.9%) was also reduced. When IVF was conducted in the straws with or without air columns, and in the microdrops, it was found that sperm penetration in the straws with air columns (96.5%) was significantly (p < 0.05) higher than that in the straws without air columns (81.7%) and in the microdrop (72.9%). However, the incidence of polyspermic penetration in the straws with air columns (34.2%) and without air columns (36.6%) was significantly (p < 0.05) lower than that (52.4%) in the microdrops. The number of spermatozoa bound to the oocytes was increased gradually in the straws but not in the microdrops in which more spermatozoa bound to the oocytes soon after insemination. CG exocytosis was more complete and faster in the oocytes inseminated in the straws than in the microdrops. These findings indicate that IVF of porcine oocytes in the straws provides a better condition in which more oocytes are fertilized normally than that in the microdrop IVF.

Effects of cooling on meiotic spindle structure and chromosome alignment within in vitro matured porcine oocytes.

Meiotic spindle structure and chromosome alignment were examined after porcine oocytes were cooled at metaphase II (M II) stage. Cumulus-oocyte complexes (COCs) collected from medium size follicles were cultured in an oocyte maturation medium at 39 degrees C, 5% CO(2) in air for 44 hr. At the end of culture, oocytes were removed from cumulus cells and cooled to 24 or 4 degrees C for 5, 30, or 120 min in a solution with or without 1.5 M dimethyl sulfoxide (DMSO). After being cooled, oocytes were either fixed immediately for examination of the meiotic spindle and chromosome alignment or returned to maturation medium at 39 degrees C for 2 hr for examination of spindle recovery. Most oocytes (65-71%) cooled to 24 degrees C showed partially depolymerized spindles but 81-92% of oocytes cooled at 4 degrees C did not have a spindle after cooling for 120 min. Quicker disassembly of spindles in the oocytes was observed at 4 degrees C than at 24 degrees C. Cooling also induced chromosome abnormality, which was indicated by dispersed chromosomes in the cytoplasm. Limited spindle recovery was observed in the oocytes cooled to both 4 and 24 degrees C regardless of cooling time. The effect of cooling on the spindle organization and chromosome alignment was not influenced by the presence of DMSO. These results indicate that the meiotic spindles in porcine M II oocytes are very sensitive to a drop in the temperature. Both spindle and chromosomes were damaged during cooling, and such damage was not reversible by incubating the oocytes after they had been cooled.

Maturation of porcine oocytes after cooling at the germinal vesicle stage.

Maturation of porcine oocytes was examined after oocytes were cooled at the germinal vesicle stage. Cumulus-oocyte complexes (COCs) collected from medium-sized follicles were cooled at 24 degrees C or 4 degrees C for 5, 30 or 120 min in a solution with or without 1.5 M dimethylsulfoxide (DMSO). After rewarming, COCs were cultured in maturation medium at 39 degrees C, 5% CO2 in air for 44 h. Meiotic spindle organisation (by immunostaining and confocal microscopy), nuclear maturation (by orcein staining) and cytoplasmic maturation (by intracellular glutathione assay) of oocytes were examined after maturation. When COCs were cooled at 24 degrees C for various times in the medium without DMSO, a tendency to decreased spindle formation, nuclear maturation and cytoplasmic maturation was observed, but there was no statistical difference compared with controls. Addition of DMSO during cooling inhibited subsequent nuclear maturation and spindle formation. When COCs were cooled at 4 degrees C, both nuclear and cytoplasmic maturation as well as spindle formation were inhibited in most oocytes in a time-dependent manner. DMSO during cooling did not have any beneficial effect on subsequent oocyte maturation and spindle formation. These results suggest that porcine oocytes are very sensitive to a drop in the temperature before exposure to culture. Cooling oocytes before maturation inhibits their subsequent spindle organisation, nuclear and cytoplasmic maturation. Addition of DMSO to the cooling solution did not protect porcine oocytes from cooling-induced damage.

Synergetic effects of epidermal growth factor and estradiol on cytoplasmic maturation of porcine oocytes.

This study was conducted to examine the effect of epidermal growth factor (EGF) and 17beta-estradiol (E2) on nuclear and cytoplasmic (male pronuclear formation and early embryo development) maturation of porcine oocytes. Oocytes were aspirated from antral follicles and cultured in modified TCM-199 medium supplemented with 0.57 mM cysteine, 10 IU/ml eCG, 10 IU/ml hCG, with or without EGF and/or E2. In vitro fertilisation of matured oocytes was performed in a modified Tris-buffered medium (mTBM) with frozen-thawed ejaculated spermatozoa. Oocytes were transferred to NCSU-23 supplemented with 0.4% bovine serum albumin at 6 h after in vitro fertilisation. Significantly higher (p < 0.05) rates of nuclear maturation, pronuclear formation and cleavage (91.7%, 65.2% and 37.3%, respectively) were observed when oocytes were cultured in the medium containing both EGF (10 ng/ml) and E2 (1 microg/ml) than in the medium supplemented with either EGF or E2 or without both. Intracellular glutathione concentration in the oocytes cultured in the medium containing both E2 and EGF was also significantly higher (12.1 pmol per oocyte) than that of oocytes cultured in the medium with E2 or EGF alone or without both. These findings suggested that EGF and E2 have a synergestic effect on both nuclear and cytoplasmic maturation of porcine oocytes.

Extracellular and intracellular factors affecting nuclear and cytoplasmic maturation of porcine oocytes collected from different sizes of follicles.

Nuclear and cytoplasmic maturation of porcine oocytes collected from different sizes of follicles were examined. Oocyte-cumulus complexes were collected from small (1-2 mm in diameter), medium (3-6 in diameter) and large (7-8 mm in diameter) follicles and cultured in a modified tissue culture medium 199 for 44 h. Nuclear maturation was evaluated after orcein staining, and cytoplasmic maturation was evaluated by intracellular glutathione (GSH) assay. Oocyte diameter, cumulus morphology, steroid hormones and glutathione in the follicular fluid (FF), were also examined. Significantly higher proportions of oocytes collected from large and medium follicles reached metaphase II than did oocytes from small follicles. Oocytes from small follicles also had a smaller size. GSH content was significantly higher (p < 0.05) in oocytes from large (14.24 +/- 2.1 pmol/oocyte) and medium (13.69 +/- 1.5 pmol/oocyte) follicles than in oocytes from small (9.44 +/- 1.28 pmol/oocyte) follicles just after collection. After maturation, oocytes from medium follicles had a higher GSH concentration than oocytes from small follicles. It was found that between 49.7 +/- 5.18 nM and 52.25 +/- 0.78 nM GSH was present in FF but there was no statistical difference between follicle sizes. A significantly higher (p < 0.001) estradiol level was present in FF from large follicles (299.2 +/- 68.6 ng/ml) than from medium (40.0 +/- 6.4 ng/ml) and small (41.2 +/- 3.7 ng/ml) follicles. Progesterone concentrations in FF from large (281.6 +/- 45.9 ng/ml) and medium (267.5 +/- 38.6 ng/ml) follicles were significantly higher than that (174.7 +/- 22.0 ng/ml) from small follicles. These results indicate that the oocyte's ability to accumulate intracellular GSH during maturation, and extracellular steroid hormones and cumulus cells, affect the competence of porcine oocytes to undergo nuclear and cytoplasmic maturation.

[Effect of angiotensin II on follicular atresia in mouse].

The effect of angiotensin II (Ang II) on the follicular development was studied by using an animal model of follicular atresia induced by pregnant mare s serum gonadotropin (PMSG). The results showed that: (1) a large number of atretic follicles were found in the ovary of 24-day-old mouse after 6-day treatment of PMSG. Deoxyribonucleic acid (DNA) extracted from granulosa cells clearly showed a ladder band under agarose gel electrophoresis analysis. (2) the contents of Ang II in the ovary extremely increased with the development of follicular atresia. (3) Ang II significantly antagonized the stimulating effect of the follicle-stimulating hormone (FSH) on estradiol (E(2)) generation of granulosa cells. It is suggested that Ang II may be involved in the regulation of follicular atresia in mouse.