The effects of different concentrations of etoposide and cycloheximide (ETO-CHXM), used for chemical enucleation of mouse oocytes, on polar body extrusion and chromatin expulsion were tested. The developmental ability of blastomeres of late 2-cell stage embryos fused to chemically enucleated oocytes of different ages or cytoplasts from different sources was also examined in vitro. Metaphase I oocytes cultured in different concentrations of ETO-CHXM (10-50 µg/ml each) extruded polar bodies at rates similar to those cultured without ETO-CHXM (58.5-65.9% and 64.6%, respectively). However, low percent of the oocytes (1.7-6.2%) expressed signs of meiotic perturbation, which was manifested by blebbing of the cytoplasmic membrane and extrusion of two or more polar body-like fragments. Twenty-three percent of the chemically enucleated oocytes cultured in ETO-CHXM-free medium spontaneously fused to their polar bodies. The rates of total chromatin expulsion were similar when ETO-CHXM concentrations were 36 and 50 µg/ml (93.5 and 98%, respectively). The results also showed that the cleavage rates of reconstituted embryos were significantly (P<0.001) affected by the age of the chemically enucleated oocytes. Cytoplasts of bisected oocytes that matured in vivo supported the development of 31.7% of the reconstituted embryos to the blastocyst stage. However, both cytoplasts of chemically enucleated oocytes and in vitro matured oocytes did not support development to the blastocyst stage. A high percentage (85.5%) of the reconstituted embryos with chemically enucleated recipients displayed abnormality of the metaphase plate. These results suggest that concentrations of etoposide between 36 and 50 µg/ml are optimum for enucleation of mouse oocytes. Furthermore, increasing the age or reducing the cytoplasmic volume of the chemically enucleated oocytes did not improve the development of the reconstituted embryos to the blastocyst stage.-KEY WORDS: chemical enucleation, cytoplast source, mouse, reconstituted embryo. chromatin could be removed or inactivated during these procedures. Thus, the quality and quantity of recipient cytoplasm vary not only from technique to technique but also from person to person. Fulka and Moor [6] recently described a novel approach to enucleate mouse oocytes chemically at metaphase I using etoposide (ETO) and cycloheximide (CHXM). The obvious advantage of chemical enucleation is in terms of the speed of the enucleation process which overcomes the difficulties of micromanipulation. However, it was reported that perturbing mitosis induces abnormal mitosis in mammalian cells [4, 12]. The same phenomena might occur during meiotic perturbation and be harmful to the oocytes. Thus, before using chemically enucleated oocytes for embryo reconstitution and nucleocytoplasmic interaction studies, further investigation and refinement of this technique is needed. The present study was designed to examine the effects of different ETO and CHXM (ETO-CHXM) concentrations on maintaining binding and enhancing chromosome exp...

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The influence of thyroid deficiency and the administration of thyroxine on pituitary-testicular function were studied in male albino rats from weaning age (22 days old) up to 82 days of age. The results showed that the hyperthyroid state induced by a daily injection of 2.5 or 5 µg L-thyroxine resulted in acceleration of growth, a comparative increase in size and number of spermatogenic and interstitial cells, an increase in the STH cells, particularly at the earlier age (42 days old), and in a decrease in the number and size of TSH cells. Gonadotrophic FSH and LH and prolactin cells exhibited an increase in their granular content. The hypothyroid state induced by thyroidectomy or thiourea feeding, at the levels of 0.1 and 0.2%, resulted in the depression of growth rate, destructive changes of the spermatogenic and interstitial cells and also in the lumen of the seminiferous tubules. A decrease in the STH, gonadotrophic FSH and LH and prolactin cells and hypertrophy of TSH cells accompanied by degranulation were also observed.

This study was carried out to test the ability of sucrose-exposed chemically enucleated mouse oocytes to support the development of reconstituted embryos in vitro. Cumulus-enclosed germinal-vesicle-stage mouse oocytes were matured in vitro to metaphase I stage and were chemically enucleated with 50 microg mL(-1) etoposide in tissue culture medium 199. The chemically enucleated oocytes were grouped into two groups. Group I was exposed to 0.75 M sucrose and group II was not exposed to sucrose. The zonae pellucidae of the chemically enucleated oocytes were removed with acid Tyrode's solution (pH 2.7). They were then aggregated into couplets with karyoplasts from pronuclear-stage embryos using phytohemagglutinin-P. The couplets were electrically fused to form reconstituted embryos. The reconstituted embryos were activated with 7% ethanol and cultured in vitro in simplex optimisation medium to test their developmental ability to the blastocyst stage. Some of the reconstituted embryos that developed to the blastocyst stage were used for chromosome counts to test their ploidy. The results of the present study showed that chemically enucleated oocytes exposed to sucrose supported the development of reconstituted embryos to the blastocyst stage (21.5%), whereas those not exposed to sucrose did not. The chromosome counts showed that the reconstituted embryos had normal ploidy (40 chromosomes). It is concluded that sucrose exposure improves the quality of chemically enucleated mouse oocytes. Thus they can be used as recipients for mouse embryo cloning and nucleocytoplasmic interaction studies.