The origin and distribution of cortical granules in human oocytes with reference to Golgi, nucleolar, and microfilament activity.

The origin and distribution of cortical granules were investigated in human preovulatory oocytes at various phases of maturation. Twenty-five oocytes obtained from unstimulated small antral follicles and from stimulated large antral and mature follicles were examined by transmission electron microscopy. Ovarian stimulation in women was accomplished by administering Clomid followed by hMG or hCG or both. Small antral follicle oocytes were dissected from ovarian biopsies, while the other oocytes were recovered by laparoscopy. Some oocytes were allowed to mature in Ham's F-10 or Whittingham's T-6 media before routine fixation in glutaraldehyde/osmium. Cortical granules originate from typical, hypertrophic Golgi complexes during early maturation and continue till its completion. Evidently there are two waves of cortical granule synthesis, the first more prolific than the second. The first occurred in small antral follicle oocytes, when there was a peak in Golgi activity, and the second was observed at the germinal vesicle stage, particularly at the onset of resumption of meiosis. Golgi complexes became progressively scarce as oocytes completed first maturation. Golgi membranes were also involved in the formation of lysosomes. A well-defined band of microfilaments was detected in small antral follicle oocytes which seemed to prevent the cortical granules, organized in a single layer, from migrating to the periphery. This band gradually became disorganized at the germinal vesicle stage as oocytes resumed meiosis, when cortical granules were apparently migrating to the surface. Metaphase I and mature oocytes had one to three discontinuous layers of cortical granules beneath the oolemma. The general organization of oocytes was also investigated and the roles of the nucleolus and endoplasmic reticulum in relation to Golgi activity and cell secretion were discussed.

EGF, TGF-alpha and EGFR expression in human preimplantation embryos.

Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha) through their common receptor, epidermal growth factor receptor (EGFR) are known to enhance mitogenesis, development and implantation in several species. In the mouse, co-culture of grouped embryos in microdrops increases the cell number and proportion developing to the blastocyst stage. A similar effect is observed with culture of single embryos in medium supplemented with EGF or TGF-alpha highlighting their embryotrophic effects. To study the role of EGF, TGF-alpha and EGFR in early human development, two methods applicable for analysis of expression at the single embryo level have been employed. In the first method, reverse transcription-polymerase chain reaction has been used to examine the presence of transcripts. Following reverse transcription, strategically designed nested primers, optimised for specificity, were used for amplification from the cDNA equivalent of a single embryo. The products were then verified by restriction enzyme digestion and sequence analysis. In the second method, immunocytochemistry has been used to colocalise the expressed proteins. Individual embryos were paraffin embedded and serial sectioned, allowing adjacent sections to be examined with different antibodies and controls. Monoclonal TGF-alpha and polyclonal EGF and EGFR primary antibodies were used. Staining was performed by peroxidase-conjugated avidin-biotin immunocytochemistry with the appropriate controls. The combination of these two methods can potentially be used for simultaneous analysis of several growth factors and/or their receptors in the same human embryos. Transcripts for EGF, TGF-alpha and EGFR were detected in unfertilized oocytes and embryos between 8-cell and blastocyst stages on day 3 to 6 post-insemination.(ABSTRACT TRUNCATED AT 250 WORDS)

The formation of oxidatively induced high-molecular-weight aggregate of alpha-/gamma-crystallins.

alpha-/gamma-Crystallin interactions under oxidation with ascorbate-FeCl3-EDTA-H2O2 followed by dialysis have been studied. A high-molecular-weight aggregate (HMWA) composed of alpha- and gamma-crystallin was observed for the mixture of the dialyzed alpha-crystallin and the oxidized gamma-crystallin through gel-filtration chromatography. This illustrates an interaction between alpha-crystallin and partially denatured gamma-crystallin induced by oxidation. No HMWA formation was observed under the condition without dialysis and/or with the addition of catalase to the oxidized gamma-crystallin prior to the addition of alpha-crystallin. More HMWA was formed by oxidized gamma-crystallin followed by the addition of alpha-crystallin than by simultaneous oxidation of both alpha- and gamma-crystallins. Conformational changes of alpha-crystallin during oxidation analyzed by circular dichroism spectra showed that oxidized alpha-crystallin can gradually be restored to an ordered structure through dialysis. The overall results imply that structural changes of both alpha- and gamma-crystallins and dialysis are required to form HMWA. The observation of this oxidatively induced chaperone/substrate complex suggests that an efficient chaperone-like protective action against oxidative insults may exist in vivo.

Synthesis and autoxidation of new tetracyclic 9H,10H-indolizino[1,2-b]indole-1-ones.

The new tetracyclic 9H,10H-indolizino[1,2-b]indole-1-one derivatives (7a-d, 7ea, 7eb) have been synthesized by modified Fischer indole synthesis from the enol ether of 2,5-dihydroxy-7-methyl-6-cyano-indolizine (3) and arylhydrazines (4a-g). Attempted N-methylation of 7a-d produced a series of autoxidized products including 10-hydroperoxy-1-methoxyindolizino[1,2-b]indole (9a-d) as the major product accompanied with methylperoxides (10a-d and 11a-d) and 2-formyl-3-(pyridine-2-yl)indole (12a, 12c) derivatives as the minor products. A plausible mechanism of the autoxidation is postulated based on the isolation of some intermediates. The reaction is thought to proceed through azaenolate/enamine intermediates following a novel type of autoxidation.

Triploid pregnancy after ICSI of frozen testicular spermatozoa into cryopreserved human oocytes: case report.

Although freezing oocytes is ethically more acceptable than cryopreservation of embryos, variable oocyte survival, fertilization rate and possible risk of increased ploidy after cryopreservation have precluded the widespread clinical application of oocyte cryopreservation in assisted reproduction techniques. We report a triploid pregnancy from intracytoplasmic sperm injection of recombinant FSH-stimulated frozen/thawed testicular spermatozoa into cryopreserved oocytes in a hormone replacement cycle. To our knowledge, this is the first report of a pregnancy where both gametes have been frozen. It illustrates the need for further research when applying new techniques in assisted reproduction.

Superovulation of mice with human menopausal gonadotropin or pure follicle-stimulating hormone in combination with human chorionic gonadotropin and the effects of oocyte aging on in vitro fertilization.

The response of female mice of F1 hybrids (CBA x C57/BL) to superovulatory doses of human menopausal gonadotropin (hMG) or pure follicle-stimulating hormone (FSH) in combination with human chorionic gonadotropin (hCG) was studied. Furthermore, the effect of oocyte aging in vivo on the subsequent rate of fertilization in vitro was also investigated. The oocytes were collected at 12, 18, and 24 hr after hCG injection and in vitro fertilization (IVF) was carried out in T6 medium. A higher proportion of animals responded to hMG stimulation (32/70) compared to pure FSH (15/66). Furthermore, hMG gave a higher oocyte recovery (454/32) than pure FSH (77/15). Fertilization rates of 57.8, 51.5, and 53.5% were obtained for the 12-, 18-, and 24-hr groups, respectively, after correction for parthenogenetic division of oocytes in the controls. No significant differences in fertilization rates were observed among the three time intervals used in recovering oocytes. However, as the degeneration and parthenogenetic division increased with the delay in collection of oocytes, 12 hr post-hCG injection was the best time to collect oocytes to obtain optimum results in in vitro fertilization.

Follicular-phase response in two clomiphene-human menopausal gonadotropin regimes for an in vitro fertilization program.

Two clomiphene-human menopausal gonadotropin regimes were assessed for our in vitro fertilization and embryo replacement (IVF and ER) program since September 1983. Clomiphene, 50 mg bd, was taken from day 2 for 5 days. Human menopausal gonadotropin (hMG) was given from day 6; for the first regime, 75 IU/day was given for the first 3 days, and for the second, 150 IU/day. The subsequent dosages were dependent on the estradiol response. There were 9 cases for the first regime and 10 cases for the second. The mean number of hMG ampoules given was 16.5 and 19.25, respectively. The number of follicles seen on ultrasound was 3.0 +/- 0.5 and 3.4 +/- 1.2 (mean +/- SD), respectively. There was no statistical difference in the estradiol response up to the day of laparoscopic ova recovery for the two regimes. However, a spontaneous luteinizing hormone (LH) surge was observed in 4 of 9 cases in the first group and 6 of 10 cases in the second group. When a comparison was made between cases that had a spontaneous LH surge and cases that were given human chorionic gonadotropin (hCG), there was a higher estradiol level on the day of the laparoscopy in the hCG group with the lower hMG regime (P less than 0.05). There were no other differences. Our small series shows a 52.6% incidence of spontaneous LH surge with clomiphene-hMG. Hence such stimulated regimes can result in a high proportion of spontaneous LH surges; this may be an index of satisfactory endocrinological control in spite of an increase in the number of follicles.