着床与生长因子的关系研究进展

论述了着床的生理机制。同时介绍了EGF、TGF、KGF和PDGF等生长因子对着床的调控作用。     

葡萄二倍体与四倍体品种间杂交胚珠的离体培养

为了克服葡萄二倍体品种与四倍体品种之间的交配障碍,进行了杂交胚珠的离体培养研究。发芽胚珠的取样时期集中于授粉后35～75d,其中多数以二倍体作母本的组合在授粉后55d取样的发芽率最高,以四倍体作母本的组合授粉后70d取样的发芽率最高。基本培养模式为:发育培养基-(剥胚)萌发培养基-生长培养基。用二倍体品种作母本时,所有组合均获得了培养苗,在1号培养基上培养的胚珠发芽率高于2号培养基。反交对培养基的要求有相反趋势,其中2个组合未得到培养苗。授粉后60d,直接剥出杂种胚进行培养,得到了最高发芽率。综合分析初步认为,用二倍体品种作母本比用四倍体品种作母本容易获得杂交后代。     

奶牛冷冻胚胎规模化移植效果研究

为了提高奶牛良种化程度,对奶牛冷冻胚胎的规模化移植效果进行了研究。结果表明:PG二次肌注法的发情率显著高于(P<0.05)PG一次肌注法,两种方法的发情同期率均超过85%,受体牛发情时间多集中在肌注PG后48～72h;胚胎解冻后的复苏率达到82.58%,且早期囊胚与囊胚的复苏率要高于桑椹胚(P>0.05);共移植受体牛218头,受胎97头,受胎率为44.5%,且春、秋季的移植效果差异不显著(P>0.05)。为奶牛冻胚的规模化移植研究提供了参考依据。     

家畜繁殖生物技术研究现状及趋势

牛胚胎移植技术已趋于成熟。我国新疆牧科院用一步细管法移植牛冻胚受胎率达41%;牛和羊鲜胚四分胚移植也产下1头犊牛和6只羔羊。家畜体外受精,因卵子体外成熟和受精卵体外培养尚不过关,目前仍停留在实验室阶段。胚胎性别鉴定,1990年Koopman发现单拷贝基因,该基因是Y染色体的性决定区,命名为SRY,可利用PCR技术制成雄性特异DNA探针盒,进行马、牛、羊、猪早期胚胎性别鉴定。北京农学院等单位用PCR扩增牛SRY序列进行奶牛胚胎性别鉴定准确率达100%。英、日、法等国已获得牛胚胎细胞核移植后代;我国也获得1只核移植兔。北农大和新疆牧科院合作以绵羊精子为载体导入牛生长激素基因rMTbGH DNA成功,外源基因整合率为3%。     

银杏幼胚离体培养再生植株的研究

 以佛手银杏幼胚为外植体, 研究了不同大小幼胚在不同培养条件下, 愈伤组织和胚状体的诱导发育情况。结果表明: 大于3 mm的幼胚, 在MK +NAA 1.0 mg·L ^{- 1} +BA 1.0 mg·L ^{- 1}培养基上胚状体诱导率最高, 达到53.6% , 最多的一块愈伤组织形成多达38个胚状体。暗培养不利于胚状体的发生。在MK培养基上添加10%椰汁对胚状体的生长发育有很好的促进作用, 有34.5%生长成苗。     

枣胚培技术体系的建立

 以‘六月鲜’枣为试材, 对花后12～82 d的幼胚进行培养。结果表明, 适宜于胚挽救的最小胚龄为花后50 d左右; 在胚形成前进行离体培养, 蔗糖浓度以7%为宜, 而胚形成后离体培养以5%蔗糖为宜; 暗培养可促进离体胚的生长发育, 且有利于生根; 植物生长调节剂及有机添加物极显著地影响着胚培效果。花后70～72 d胚龄的胚适宜培养基为1 /2MS + IBA 0.4 (mg/L, 单位下同) +BA 0.8 +LH 500 + GA₃5 + 5%蔗糖。胚培苗经扩繁后转入1 /2 MS + IBA 1.0 + IAA 0.06 + 2%蔗糖培养基上, 获得了健壮生根苗,生根苗移栽成活率达85%以上。     

Quick detection of sex determining region protein gene in mouse fetuses

AIM: To detect quickly the Y-chromosome specific sex determining region protein (Sry) gene in mouse fetuses on embryonic day 14.5 with a PCR method. METHODS: We designed specific primers with the OLIGO 5. 0 software. Templates were prepared in 30 minutes by the following way. About 1 mg embryonic tissue but not fetal liver was suspended, and treated with 200μL of lysis buffer, consisting of PCR buffer containing 20 mg/L proteinase K, 0. 5% NP-40, and 0.05% Tween 40, at 60°C for 15 minutes, heated for 5 minutes at 100 °C, 10μL was used as template. The PCR react ion was performed in 50μL, using two sets of primers specific for Sry gene (chromosome Y) and IL-3 gene (chromosome 11) . PCR conditions and cycle numbers were optimized. The assessment of the results was done by electrophoresis in 3% agarose run at high voltage. The specificity of the method was conf irmed by fluorescent in situ hybridization (FISH) using a specific male probe on embryonic tissue cells. RESULTS: Electrophoresis showed that PCR product of male control DNA consisted of a 649 bp product representing the IL-3 gene and a 444 bp product representing the Y-specific Sry gene, female control DNA only one 649 bp product. Fetuses with two bands matching those as seen inmale control DNA are the presumpt ive male fetuses. Fetuses, only the IL-3-associated 649 bp band, are the presumptive female fetuses. These were confirmed by FISH. The ent ire procedure took <3. 5 h. CONCLUSION: The established PCR assay offers a quick, simple, accurate, and sensitive detection of sex determining region protein gene in mouse fetuses. This method allowed the preparation and culture of pure male and female hematopoietic stem cells from fetal tissue.     

Study on the electrophysiological characteristics of ion channels of stem cell derived cardiomyocytes of mouse

AIM: To study the electrophysiological characteristics of ion channels of stem cell derived cardiomyocytes(SCDC) of mouse. METHODS: Embryonic stem cells of D₃ line(ES-D₃) were cultured on the MEF feeder layer with BRL conditioned medium, and fetal mouse heart cells(FMHC)were cultured in vitro. Then ES-D₃ cells were induced to differentiate into many kinds of cells. SCDC were harvested on day 12 after differentiation initiating and identified by electro-microscope and immunocytochemistry. SCDC and FMHC were prepared for the patch-clamp research. Sodium and calcium currents together were elicited and compared between SCDC and FMHC. RESULTS: The current characteristics of sodium and calcium channels of SCDC were very similar to FMHC. CONCLUSION: The functional expression of ion channels occurred during ES-D₃ cells differentiation and the electrophysiological characteristics of sodium and calcium channels of SCDC are very similar to FMHC.     

Differential gene expression of Eph‐ephrin A1 and LEPR‐LEP with high or low number of embryos in pigs during implantation

The objective of this study was to ascertain whether mRNA and protein expressions of implantation‐related genes (erythropoietin‐producing hepatocellular receptor–ligand A1, Eph‐ephrin A1 and leptin receptor–leptin, LEPR‐LEP) differed between pigs with high and low number of embryos, and whether these differences in gene expression might affect embryo implantation. Experimental pig groups (n = 24) for high and low number of embryos were prepared by altering the number of eggs ovulated in pre‐pubertal gilts treated with 1.5 × (High) or 1.0 × (Low) PG600 ([400 IU PMSG + 200 IU hCG]/dose, AKZO‐NOBEL). Gilts expressing oestrus were artificially inseminated twice and maintained in breeding and gestation until the reproductive tract was collected on day 22 of pregnancy. At slaughter, the reproductive tracts from each pregnant gilt from each treatment were immediately processed to collect samples for RNA and protein analysis. Within each gilt, three conceptus points were sampled, one from each horn and then a random conceptus within the tract. At each conceptus point, endometrial attachment site, chorion–allantois and embryo were collected and immediately frozen in liquid nitrogen. Number of corpus luteum (CL) (35.4 vs. 12.6) and total embryo number (18.8 vs. 10.2) were greater in the high‐embryo compared to the low‐embryo group, respectively (p < .05). Real‐time qPCR results showed that Eph‐ephrin A1 mRNA expression was less in the high‐embryo (p < .05) compared to the low‐embryo group. In addition, Western blotting analysis indicated that Eph‐ephrin A1 and LEP protein expression at endometrial attachment site in high‐embryo was less (p < .05) compared to low‐embryo group. It was also noted that mRNA expression of Eph‐ephrin A1 and LEPR‐LEP was greater in pregnant than non‐pregnant gilts (p < .05). Moreover, mRNA expression of Eph‐ephrin A1 (p < .05) and LEPR‐LEP was greatest at endometrial attachment site among all three tissues. There was a positive correlation between expressions of Eph‐ephrin A1, LEPR‐LEP and embryo length with the correlation coefficient 0.31–0.59. For Eph‐ephrin A1, the highest correlation coefficient appeared between Eph A1 expression and normal embryo number, between ephrin A1 expression and embryo length. For LEPR‐LEP, the highest correlation coefficient appeared between LEPR‐LEP expression and ovary weight (0.79 for both, p < .05), followed by embryo length and weight. The results of this study suggest that low expression of Eph‐ephrin A1 and LEPR‐LEP is somehow related to increased embryo number during implantation and that endometrial attachment site might be the main target tissue of these gene products. Yet, the increased expression of Eph‐ephrin A1 and LEPR‐LEP appeared associated with increased embryo growth (length and weight) and ovary weight, Eph‐ephrin A1 and LEPR‐LEP might play roles in the regulation of embryo implantation in pigs.     

Factors Affecting Recipients' Pregnancy,Pregnancy Loss,and Foaling Rates in a Commercial Equine Embryo Transfer Program

During 11 breeding seasons, 351 7- to 10-day-old horse embryos were nonsurgically transferred into recipients that ovulated between 3 and 10 days earlier. Pregnancy rates at 14 and 40 days and foaling rates were 77.8% (273/351), 69.2% (243/351), and 64.4% (226/351), respectively. Pregnancy loss between 14 and 40 days was 11% and between 40 days and delivery was 7%. The transfer of quality grade 3 to 4 embryos resulted in a significantly lower pregnancy rate at 14 days compared with the transfer of grade 1 to 2 embryos (46.2% vs. 79%; P < .05). Eight-day-old embryos resulted in significantly lower pregnancy losses than day 9 or 10 embryos, as occurred for embryos between 400 and 1200 μm compared with embryos <400 μm. Embryos recovered from mares >20 years resulted in a significantly higher pregnancy loss rate than those recovered from younger mares. The same happened for embryos coming from mares affected by reproductive pathologies compared with healthy mares performing sport activity. None of the evaluated parameters influenced recipients' foaling rate significantly.