毫米波辐射对植入前小鼠胚胎及早期胚胎的影响

本文报道小鼠受精卵体外及在体受毫米波辐照后一些改变。毫米波源为36.11GHz固态微波连续发生器,波长8mm,功率密度为10mW/cm~2、8mW/cm~2、4mW/cm~2及2mW/cm~2。结果发现2-4mW/cm~2毫米波辐照即可使体外培养之受精卵细胞表面微绒毛减少、脱落,细胞表面形成许多囊泡。透射电镜下可见细胞间隙扩大、胞浆中线粒体膨胀、空化,用FITC-ConA试验可见细胞表面结合荧光减少。酶试验证明辐照后卵胚细胞表面AKP,ATPase,5′-Nase均有降低。在体受精卵细胞经辐射后证明,辐射可使胎儿体重增长减慢,囊胚形成数量下降,植入率降低,而表面酶下降不明显。     

Fertilization and early embryology: The protective action of polyvinylpyrrolidone-Percoll during the cryopreservation of mouse 2-cell embryos and its effect on subsequent developmental potential post-thaw in vitro and in vivo

The effects of cryopreservation, in media containing (FS3 +) or omitting (FS3) polyvinylpyrrolidone (PVP) in the form ofPercoll (PVP-Percoll), on the survival of 2-cell mouse embryoswas studied. Survival and zona pellucida disruption post-thaw,growth (assessed by in-vitro culture until the blastocyst stage)and development in vivo (assessed by implantation and livingfetus rates and the birth of live progeny) were all investigated.Initial post-thaw survival showed no statistically significantdifference (P 0.05) between FS3+ (91.1 ± 9.8%) and FS3(84.5 ± 6.6%). However, there was a statistically significant(P 0.05) reduction in the incidence of zona damage when thefreezing solution contained PVP-Percoll compared to the control(3.6 ± 1.0 and 8.7 ± 0.6% respectively) and astatistically significant (P 0.05) greater number of embryosdeveloping in vitro to the blastocyst stage (84.8 ±7.1and 72.3 ± 6.1% respectively). The rates of implantationwere not significantly different: 72.2 ± 7.0% for FS3+and 51.2 ± 30.7% for the non-frozen control group. Thepercentage of live fetuses was also similar between the experimentaland control groups: 27.4 ± 10.6 and 24.3 ± 113%respectively. We conclude that the presence of polymers canprotect embryos against cryoinjury and that PVP in the formof PVP-Percoll provides a non-toxic alternative to PVP in itsnative form, during the cryopreservation of mouse 2-cell embryos.     

Immunocytochemistry of ambiguous cells in adult and embryonic dwarf (dw) mouse pituitaries

In the pars distalis of the pituitary gland in adult and embryonic dwarf (dw/dw) mutant mice, ambiguous cells exhibiting ultrastructural features common to growth hormone (GH) cells and prolactin (Prl) cells were analyzed by means of colloidal gold ultrastructural immunocytochemistry in order to define the functional nature of these peculiar cells. Adult and 18-day embryonic pituitaries from normal (+/+; dw/+) and dwarf (dw/dw) mice were processed with antibodies to GH, Prl, TSH (thyroid-stimulating hormone), ACTH (adrenocorticotropic hormone), LH (luteinizing hormone), FSH (follicle-stimulating hormone), and HCG (chorionic gonadotropic hormone). In the adult and embryonic dwarf pituitaries, the ambiguous cells reacted negatively to all of the antibodies except for anti-ACTH, which labeled them well. In addition, the ACTH-positive cells showed a much wider variety of shapes and granule size and distribution, as compared with normal adults. In the embryos, this variability in ACTH cell morphology occurred not only in dwarf embryos, but in their normal counterparts as well. The results thus suggest that adult dwarf pituitaries may retain an embryonic or incompletely differentiated form of ACTH cells. © 1993 Wiley-Liss, Inc.     

生长因子作为评价胚胎质量的生物学标记物的研究

目的　研究表皮生长因子 (epidermalgrowthfactor,EGF)、转化生长因子 - β1(transforminggrowthfactor- β1,TGF -β1)、胰岛素样生长因子 -Ⅱ (insulin -likegrowthfactor-Ⅱ ,IGF -Ⅱ )做为评价小鼠胚胎质量生物学标志物的可能性。方法　取小鼠二细胞胚胎 ,分为正常和异常两组 ,体外培养至囊胚期 ,对胚胎的生长发育及着床能力进行形态学观测 ;检测胚胎培养液中三种生长因子的含量 ,研究生长因子含量与胚胎质量间的关系。结果　 1.正常组胚胎培养液中EGF、IGF -Ⅱ的含量显著高于异常组 ,且EGF含量与胚胎生长发育的形态学指标呈正相关 ,但与着床指标相关不显著 ;2 .IGF -Ⅱ与胚胎生长发育及着床指标均呈正相关。 3.TGF - β1的含量两组间差异无显著性。结论　胚胎培养液中EGF、IGF -Ⅱ含量测定可反映胚胎生长发育能力 ,IGF -Ⅱ还可反映胚胎着床能力 ,从而有利于了解胚胎功能状态及生存潜力 ,为胚胎质量的检测提供了一条新的思路     

Recovery, survival and functional evaluation by transplantation of frozen-thawed mouse germ cells

BACKGROUND: Establishing a successful method for testicular stem cell transplantation of frozen-thawed testicular cells would be of immense benefit to boys with childhood cancer undergoing a sterilizing treatment. In this study, we evaluated different cryopreservation protocols in a mouse model by means of testicular germ cell transplantation (TGCT), in order to establish an optimal freezing protocol. METHODS AND RESULTS: In a first series of experiments, we compared an uncontrolled protocol with 1.5 mol/l dimethyl sulphoxide (DMSO) versus a controlled long protocol (cooling to -80 degrees C) and observed a better viability with the latter protocol (36% versus 48%, P < 0.05). We then compared survival after two thawing methods (37 degrees C water versus ice water) in either a DMSO- or an ethylene glycol (EG)-based protocol, and found no difference. In order to evaluate the functional capacity of the cryopreserved testicular suspension, TGCT was performed with both fresh and frozen-thawed suspensions. In 90% of the successfully injected testes, spermatogenesis was reinitiated using fresh suspensions. In contrast, this figure was only 12.5 and 22.7% after cryopreservation, for the short controlled EG protocol and the uncontrolled DMSO protocol, respectively. CONCLUSION: Reinitiation of spermatogenesis is possible after cryopreservation of testicular germ cell suspensions. Although cell survival was acceptable, our results after TGCT show that our protocols need further improvement.     

Characterization of a whole-cell Ca2+-blockable monovalent cation current in isolated ectodermal cells of chick embryo

The presence of a Ca²⁺-blockable monovalent cation current is demonstrated in isolated ectodermal cells of the chick embryo using the whole-cell patch-clamp method. In the absence of any stimulation, the whole-cell current is time independent and rectifies outwardly at membrane potentials higher than +40 mV The outward current is neither carried by Cl^– channels nor by K⁺ channels. Application of a Ca²⁺-free solution containing 1 mmol/l ethylenediaminetetraacetic acid (EDTA) elicits a large inward current and increases the outward current. The inward current can be carried by extracellular Li⁺, Na⁺, K⁺ and Cs⁺, but notN-methyl-d-glucamine. The Ca²⁺-blockable monovalent cation channel discriminates very poorly among these cations. The estimated number of channels per cell is around 2000. Extracellular protons block the inward Na⁺ current in the absence of extracellular Ca²⁺. The apparent negative logarithm of the dissociation constant for proton (pK _H) at –100 mV is 5.8. Among 12 potential channel modulators, including verapamil and nifedipine, only quinine decreases the current. Quinine blocks this current with a dissociation constant,K _d, equal to 0.18 mmol/l, independent of the membrane potential. This study demonstrates the presence of a whole-cell Ca²⁺-blockade monovalent cation current in dissociated chick ectodermal cells with permeation properties similar to those observed at the single-channel level. Contrary to studies made of other tissues, we did not observe any blocking effect of verapamil and nifedipine on the Ca²⁺-blockable monovalent cation current.     

恒河猴tPA基因的克隆、测序与真核表达

本试验采用高浓度乙二醇（EG-8）作为胚胎冷冻保护剂,以高浓度的半乳糖作为解冻稀释液,对MMTV-Wnt-1转基因小鼠的早期囊胚进行玻璃化冷冻保存。结果在EG-2中平衡4-6min和解冻时间为3-5min时获得了最佳冷冻效果。此条件下冷冻胚胎的总回收率为88.61%（109/123）;回收胚胎在体外发育率达88.99%（97/109）;孵化率达81.65%（89/109）;发育胚胎的移植出生率为38.36%（69/181）;冷漠胚胎移植出生后经PCR检测一半小鼠整合阳性。结果表明使用高浓度的乙二醇作为冷冻保护剂和以高浓度的半乳糖作为冻稀释液能有效地保存转基因小鼠胚胎。     

Biofluid Aspects of Embryo Transfer

Embryo transfer (ET) is the last stage of extracorporal fertilization during which the embryo is placed in the uterine cavity with a medium-filled catheter 2–3 days after in vitro fertilization. While fertilization in the laboratory occurs at very high rates (>:90%), the overall success of the procedure (i.e., take home baby) is still very low (<25%) and assumed to be mainly due to implantation failure. A computational model was developed to simulate ET within the uterine cavity by a fluid-filled catheter inserted into a two-dimensional channel with oscillating walls. The results showed that the speed at which the embryos are injected from the catheter dominates the procedure and controls the velocity of their transport within the uterine cavity. ET at excessively high injection speeds may lead to ectopic pregnancies, while uterine peristalsis affects transverse dispersion only during injection at low injection speeds. The presence of the catheter within the uterus does not affect flow patterns downstream of its tip. The potential risks to implantation failure due to mechanical factors involved in the ET processes are discussed. © 2003 Biomedical Engineering Society. PAC2003: 8719-j, 8710+e     

Efficacy of intracytoplasmic sperm injection using intentionally cryopreserved epididymal spermatozoa

Microsurgical epididymal sperm aspiration was a great advancein the therapy of patients with non-recon-structable, obstructiveazoospermia, most notably congenital bilateral absence of thevas deferens. Using conventional in-vitro fertilization, pregnancieswere rarely achieved because the rate of oocyte fertilizationwas extremely poor. However, the use of retrieved spermatozoain conjunction with intracytoplasmic sperm injection (ICSI)has dramatically increased the likelihood of embryo formation.Typically, sperm and oocyte harvesting are performed simultaneously.We have investigated whether frozen-thawed spermatozoa workas well as fresh spermatozoa. When we had concluded from ourown population of patients (groups I and II) that they did,we adopted a policy of aspirating spermatozoa, primarily cryopreservingthem and using them for ICSI at a later date. We found the fertilizationrates of this latter cohort of patients (group III) to be excellent(37% per oocyte), and the ongoing pregnancy rate is quite satisfactory(40 % per couple, 29% per cycle). We offer this approach asan alternative to the traditional scheme because it markedlyeases the burden of partner scheduling on both the couple andthe clinicians involved. In addition, assurance of the availabilityof male partner spermatozoa can be attained prior to beginningovulation induction.