免疫荧光法在研究人卵母细胞、受精卵及胚胎结构和功能中的应用

目的寻找适合研究人类生殖的免疫荧光方法。方法对蛋白激酶C(PKC)在人卵母细胞、受精卵、种植前胚胎及皮质颗粒进行免疫荧光染色。结果激光共聚集显微镜可以显示PKC在人GV、MI和MⅡ期卵母细胞及受精卵中的位置,并随着细胞的不同时期分布不同。在多精受精的胚胎中,免疫荧光显微镜可以清楚地显示PKC及细胞核在卵裂球的位置。使用A23187处理卵母细胞后,激光共聚集显微镜从表面可以观察到呈新月型点状分布的皮质颗粒。结论通过改进的免疫荧光法可以获得理想的实验结果。     

Fertilization and pregnancy using metaphase I oocytes in an intracytoplasmic sperm injection program

Purpose: In this study we investigated whether metaphase I oocytes collected in an intracytoplasmic sperm injection program could successfully be matured and fertilized by injecting aged (>20-hr) spermatozoa. Materials and Methods: Metaphase I oocytes aspirated were preincubated for 20 hr to allow the oocytes to reach meiotic maturity. Only metaphase II oocytes were injected. The original sperm sample processed on the day of aspiration was used in the microinjection process. Results: One hundred eighty-three oocytes were collected, of which 42 (23%) were metaphase I oocytes. These were incubated for 20 hr and microinjected with the original sperm sample. Thirty-one (74%) of the metaphase I oocytes reached meiotic maturity (extruded polar body); 67.7% showed two pronuclei 18 hr after injection and 61.3% embryo development 40 hr postinjection. No difference in fertilization and embryo development rate was found in metaphase II oocytes injected 6 hr postaspiration versus 20 hr postaspiration. An ongoing pregnancy was also achieved using only embryos obtained from matured metaphase I oocytes. Conclusions: Metaphase I oocytes can be successfully maturedin vitro and injected using aged (>20-hr) sperm samples. Matured metaphase I oocytes, if successfully injected, produce embryos able to induce pregnancy.     

Chromosome investigation in in vitro fertilization failure

Purpose A chromosomal complement of 227 human oocytes was studied to provide information on the frequency and type of chromosomal abnormalities in oocytes failing in vitro fertilization.Results Normal haploid chromosome complement was found in 54.6%; chromosomal abnormalities consisting of diploid sets were identified in 16.7% and aneuploidy was observed in 26%. Premature condensation of sperm chromosomes of the G1-phase was observed in 22.9% oocytes. Male infertility was correlated with an increase in the rate of aneuploidy when compared with tubal infertility. The rate of chromosome abnormalities for the oocytes recovered from women who had no fertilized oocytes was significantly higher compared to those with at least one oocyte fertilized.Conclusion A high frequency of chromosome abnormalities in unfertilized oocytes suggests that natural selection against chromosome abnormalities may occur even prior to fertilization.     

Retrieval, Maturation, and Fertilization of Immature Oocytes Obtained from Unstimulated Patients with Polycystic Ovary Syndrome

Purpose: Our purpose was to determine whether immature oocytes could be retrieved under local anesthesia, whether these oocytes would mature and fertilize in vitro, and whether adequate endometrium development could be obtained after hormonal supplementation. Methods: Ovum pick-up was performed under local anesthesia. Immature oocytes were cultured and inseminated. To prepare the endometrium, estradiolvalerate was administered in combination with micronized progesterone. Results: Immature oocytes were obtained in all cases. Fifty-six percent (n = 30) of the oocytes developed into metaphase II (MII) after 48 hr of culture, and another 20% reached the MII stage by 72 hr. Normal fertilization was observed in only 10% of oocytes inseminated. No embryonic development occurred, and therefore embryo transfer was not performed in any of the patients. Endometrial microbiopsy was performed in all subjects and endometrial development was considered sufficient in eight patients. Conclusions: We collected immature oocytes from patients with polycystic ovary syndrome without general anesthesia. In vitro maturation of these oocytes seemed adequate but fertilization rates were poor. Sufficient endometrial quality was obtained after hormonal substitution.     

Radioligand binding affinity and biological activity of the enantiomers of a chiral melatonin analogue

Melatonin, a hormone secreted by the pineal gland, can act on the central circadian oscillator in the suprachiasmatic nucleus of the hypothalamus. It has been proposed that melatonin or its analogues may be useful in restoring disturbed circadian rhythms in jet-lag, shift-work and some blind subjects, and as sleep-promoting agents. In the present study, the (−)- and (+)-enantiomers of N-acetyl-4-aminomethyl-6-methoxy-9-methyl-1,2,3,4-tetrahydrocarbazole (AMMTC) were separated and tested. The affinity of the enantiomers at the specific 2-[¹²⁵I]iodomelatonin binding site in chick brain membranes was compared in competition assays, and their biological activity in a specific melatonin receptor bioassay, aggregation of pigment granules in Xenopus laevis melanophores. The (−)-enantiomer of AMMTC was 130-fold and 230-fold more potent than the (+)-enantiomer in competition radioligand binding assays and melanophores, respectively. Both enantiomers are melatonin receptor agonists; (−)-AMMTC is slightly more potent than melatonin itself. As the tetrahydrocarbazole nucleus holds the C-3 amido side-chain of AMMTC in a restricted conformation, the analogues will be useful in modelling the melatonin receptor binding site.     

The YAG laser used in micromanipulation to transect the zona pellucida of hamster oocytes

Problem Since there has been no reported use of the YAG laser to micromanipulate oocytes, our purpose was to study whether (1) a YAG laser could be used to open the zona pellucida of hamster oocytes; (2) human sperm could reach the ooplasm and (3) under sperm penetration assay conditions, sperm would bind and penetrate the ooplasm.Results A YAG 100 laser was used at 10 W and 0.4-sec pulse width to open eight of eight ooplasm oocytes. The opening in the zonae was 0.25 to 1.0 rad (10 to 40 µm). For the initial eight oocytes and two parallel controls, the coarse appearance of the ooplasm was unchanged after 3 days. Next, in 11 of 12 manipulated oocytes, the sperm clustered at the opening of the zona. When 16 more oocytes were opened and exposed to sperm in sperm penetration assay conditions, each ooplasm bound sperm. There was no penetration noted. Each manipulation time was <1 min. To clarify the laser effect, oocytes were exposed to laser energy then utilized as the interactive surface in the sperm penetration assay. It was found that only 20% bound sperm with no penetration. Conclusion While the time factor compares favorably with other methods of zona opening, further study needs to be performed to minimize effect to the exposed oocyte.     

Female Germ Cell Loss From Radiation and Chemical Exposures

Female germ cell in some mammals are extremely sensitive to killing by ionizing radiation, especially during development. Primordial oocytes in juvenile mice have an LD₃₀ of only 6-7 rad, and the germ cell pool in squirrel monkeys is destroyed by prenatal exposure of 0.7 rad/day. Sensitivity varies greatly with species and germ cell stage. Unusually high sensitivity has not been found in macaques and may not occur in man, but this has not been established for all developmental stages. The exquisite oocyte radiosensitivity in mice apparently reflects vulnerability of the plasma membrane, not DNA, which may have implications fur estimating human genetic risks. Germ cells can be killed also by chemicals. Such oocyte loss, with similarities to radiation effects, is under increasing study, including chemotherapy observations in women. More than 75 compounds have been tested in mice, with in vivo toxicity quantified by oocyte loss; certain chemicals apparently act on the membrane.