猪冷冻附睾精子与体外成熟卵母细胞的体外受精

研究了不同解冻温度 (39～ 41℃、5 8～ 6 0℃、6 9～ 71℃ )和解冻方式 (干解冻、湿解冻 )对猪冷冻附睾精子解冻后的活力、存活指数以及受精能力的影响。结果表明 ,采用同一种解冻方式 ,经高温解冻的精液 ,虽然解冻后的活力较好 ,但活力却下降很快 ,精子的存活时间和存活指数均不如低温解冻的精液。采用同一种解冻温度 ,湿解冻所需的解冻时间相对短 ,解冻后精子活力相对低 ,但存活时间和存活指数相应优于对应的干解冻试验组。经低温解冻的精子 ,受精能力最强 ,精子的受精能力与解冻时间呈显著正相关 (r=0 .916 13,P=0 .0 10 3)。使用改良 TCM199培养液 。

胰岛素样生长因子Ⅱ对人颗粒细胞分泌功能的影响

目的:探讨胰岛素样生长因子Ⅱ(IGF-Ⅱ)对人颗粒细胞分泌功能的影响。方法:收集行体外受精-胚胎移植(IVF-ET)患者取卵时的颗粒细胞作体外培养,在有或无尿促性腺激素(hMG)作用下,以不同浓度的基因重组人胰岛素样生长因子Ⅱ(rhIGF-Ⅱ)作用于颗粒细胞,48h后收集培养液测定雌二醇(E2)、孕酮(P),观察rhIGF-Ⅱ对体外培养的颗粒细胞分泌E2、P的影响。结果:在无hMG作用下,rhIGF-Ⅱ能够刺激颗粒细胞分泌E2增加(P<0.05);加入hMG后,rhIGF-Ⅱ与hMG协同作用能够显著增加颗粒细胞分泌E2的量(P<0.05);但不论有无hMG,rhIGF-Ⅱ对P分泌的影响均不明显(P>0.05)。结论:IGF-Ⅱ可单独或协同hMG刺激颗粒细胞甾体激素的分泌。

人类辅助生殖衍生技术

辅助生殖技术为千万不孕症家庭带来了福音,是现代医学治疗不育症的手段之一,是生殖医学领域的一场革命。近年来,一系列辅助生殖衍生技术,如卵子体外成熟、单精子卵细胞浆内显微授精术、移植前遗传学诊断、卵子、胚胎及卵巢组织冷冻技术等获得了较大的发展,为降低常规体外受精-胚胎移植的费用、风险和提高成功率等提供了可能。

Improvement of in vitro Cytoplasmic Maturation of Denuded Porcine Oocytes by Coculture with Follicular Mural Granulosa Cells

[Objective] This study aimed to improve the in vitro maturation quality of denuded porcine oocytes and provide scientific basis for establishing a stable and efficient denuded oocyte culture system. [Method] The first polar body extrusion rate, oocyte glutathione （GSH） content, positive rate of brilliant cresyl blue （BCB） staining and development potential of activated oocytes or fertilized oocytes were employed as main indicators to investigate the effects of follicular mural granulosa cell （MGC） coculture on cytoplasmic maturation of cumulus cell-removal oocytes （Denuded Oocyte, DO）. [Result] According to in vitro maturation results, compared with DO group, the first polar body extrusion rate of porcine oocytes in DO＋MGC group was not significantly different, but the nuclear maturation process was improved and was more similar to that in COC （cumulus-oocyte complex） group. Detection of GSH content in mature oocytes showed that there was no significant difference between DO＋ MGC group （optical density of 1 053.67） and COC group （optical density of 1 426.00） or between DO＋MGC group and COC＋GC group （optical density of 1 541.00）, however, GSH content in mature oocytes of DO group （optical density of 724.67） was significantly lower than that of COC group and COC＋GC group （P0.05）. Detection of glucose-6-phosphate dehydrogenase （G6PDH） activity showed that there was no significant difference in BCB positive oocyte rate between DO ＋MGC group （88.26% ） and COC group （92.75%） or between DO＋MGC group and DO group （82.86% ）, however, BCB positive oocyte rate of DO group was significantly lower than that of COC group （P0.05）. Furthermore, the cleavage rate and blastocyst rate of activated mature oocytes derived from DO ＋MGC group （94.98% and 43.67% , respectively） were significantly higher than those from DO group （52.54% and 8.97%, respectively） （P0.05）, and were not significantly different compared with those from COC group （97.11% and 38.30%, respectively）. In addition, the cleavage rate of fertilized oocytes derived from DO＋MGC group （72.65%） showed no significant difference compared with that from DO group （63.59%）, but the blastocyst rate of DO＋MGC group was significantly higher than that of DO group （9.88%） （P0.05）. [Conclusion] MGC coculture can significantly improve the in vitro cytoplasmic maturation quality of denuded porcine oocytes, thereby enhancing the subsequent developmental potential.

In vitro Maturation of Tan Sheep Oocytes

[Objective] This study aimed to investigate the appropriate concentrations of follicle-stimulating hormone（FSH）, luteotropic hormone（LH） and estrodiol（E2） during in vitro maturation of Tan sheep oocytes. [Method] Tan sheep oocytes were divided into five groups for in vitro maturation culture： control group, FSH group（10,50, 100, 200 and 300 μg/ml FSH, respectively）, LH group（5, 10, 20, 50 and 100μg/ml LH, respectively）, E2group（5, 10, 25, 50 and 100 μg/ml E2, respectively）, and FSH ＋ LH group（100 μg/ml FSH ＋ 20 μg/ml LH）. The releasing rate of first polar bodies was analyzed. [Result] The maturation rate of Tan sheep oocytes in 100 μg/ml FSH ＋ 20 μg/ml LH group reached the highest（64.64%）, which was significantly higher than that in other four groups（P〈0.05）; among different FSH concentrations,100 μg/ml FSH was superior to other four concentrations and the control group, exhibiting significant differences（P〈0.05）; among different LH concentrations, 20 μg/ml LH was superior to other four concentrations and the control group, exhibiting significant differences（P〈0.05）; among different E2 concentrations, 50 μg/ml E2 was superior to other four concentrations and the control group, exhibiting significant differences（P〈0.05）. [Conclusion] Under the experimental conditions, 100 μg/ml FSH ＋20 μg/ml LH was the most appropriate hormone combination for in vitro maturation of Tan sheep oocytes.

Chronological and Morphological Progression of Nucleus during Mouse Oocyte Maturation and Fertilization in vitro

The chronological and morphological changes of the nucleus during mouse oocyte maturation and fertilization were systematically studied. Although most oocytes went through GVBD 2-4 hrs after culture, 13.6% remained at GV stage 8 hrs after culture.TEM observation revealed that nucleoli of oocytes which failed to go through GVBD were composed of fibrillar-granular component,small vacuoles and fibrillar centers or showed small vacuoles on nuclear surface. During GVBD, the nucleoli became smaller and smaller and finally disappeared with the nuclear-associated chromatin dislocated to the periphery. Nuclear membrane with attached chromatin became folded and electronic dense cores appeared in the center of chromatin clumps at the same time.The last event of GVBD was the disruption of nuclear membrane.At the end of the 5th hr after culture, meiosis progressed to prometaphase I.Chromosomes,distributed in the original GV area free of organelles,were surrounded by large quantity of mitochondria and small SER vesicles. At the end of the 12th hr after culture,48. 1% of the oocytes emitted PB1.Decondensing sperm head and early male pronuclcus(mPN)with condensed nucleoli were found 1-2 hrs after insemination.The formation and enlargement of female PN(fPN) occurred a little earlier than that of mPN. 33.3% finished syngamy at 8-9 hrs after insemination.The process of nucleolus formation was reverse to that in GVBD. The oolemma modification caused by cortical reaction could effectively inhibit polyspermy.in contrast,there were sperm binding to the oolemma where CGs failed to be released. In addition, PB2 was emitted 2-5 hrs after insemination. The difference between PB1 and PB2 as well as the abstriction of polar body were also discussed.

In vitro Oocyte Maturation in the Zebrafish Brachydanio rerio and Fertilization and Development of the Mature Egg

Maturation process of zebrafish oocyte was investigated using in vitro incubation.In medium EM-199 containing 0.5 μg/ml of 17α-hydroxyprogesterone incubated under 80％ O_2 and at 25°C,germinal vesicles(GV)of oocytes in stage Ⅳ migrated from midway between the center and theperiphery ofoocytes to the periphery in 40 minutes and the oocytes went into stage V.Half an hourlater,the oocytes underwent germinel vesicle breakdown(GVBD)with a breakdown rate of 59％.Two more hours were needed for such oocytes to complete their final maturation.The mature eggscould not come off from the follicle layer surrounding them by themselves(ovulation).By removingthe follicle and adding active sperms for insemination,we could make the mature eggs fertilized.Thechorion was elevated and blastoderm formed on the animal pole.The cleavage and development ofthese fertilized eggs followed the same course as the naturally matured and fertilized eggs.Usingblastula formation as a marker of successful fertilization of the in vitro matured egg,the fertilizationrate was 78％.This is the first report on the successful in vitro incubation of mature oocytes inzebrafish.The establishment of this in vitro oocyte maturation technology has laid the foundationfor further investigation of the transfer of foreign genes in the germinal vesicles of oocytes.