Evidence for polar cytoplasm/nuage in rat oocytes.

In many organisms oocytes contain dark-staining material, termed nuage, that is concentrated at one pole of the oocyte cytoplasm and that influences the further development of the oocyte after fertilization. In mammalian oocytes, ultrastructural studies have detected small patches of nuage-like material, but thus far no nuage-rich zone of polar cytoplasm has been reported. Here, we report that when large sections of rat ovary embedded in methacrylate resin are stained with toluidine blue and surveyed, many oocytes contain a narrow, sharply defined, basophilic zone of polar cytoplasm that appears analogous to the polar cytoplasm of Xenopus and other non-mammalian species. This basophilic polar cytoplasm was common in multilaminar follicles and was not visible in smaller, primordial follicles. In one out of five oocytes stimulated with hCG to complete the first meiotic division, a relatively faint region of cortical basophilia was detectable. Further studies will be needed to ascertain if this nuage-like material has an influence upon the development of oocytes similar to that seen in non-mammalian species.

Meiotic maturation in cultured bovine oocytes is accompanied by remodeling of the cumulus cell cytoskeleton.

Mammalian oocyte maturation and cumulus expansion are hormone-induced contemporaneous processes that involve changes in cumulus cell shape and in the interactions between cumulus cells and the oocyte. Laser scanning confocal and conventional fluorescence microscopy have been used to study the relationship between reorganization of the cumulus cell cytoskeleton and meiotic progression in bovine cumulus oocyte complexes (COCs) matured in vitro. Inclusion of hormone supplements (FSH, LH, E2) in the maturation media was required for meiotic progression to metaphase 2 and maximal cumulus expansion. Under these conditions, stage-specific changes in the cytoskeletal content and organization of cumulus cell transzonal processes was observed. Specifically, abundant, actin-filled transzonal processes were observed in GV stage COCs. In stages intermediate in the maturation process (10 hr culture), fewer actin-filled transzonal processes were seen and microtubule-filled transzonal processes became apparent. At the end of maturation (24 hr culture), numerous, elongate microtubule rich transzonal processes were evident and actin-filled processes were rarely observed. Additionally, a spatial relationship between microtubule rich transzonal processes and oocyte chromatin was consistently observed in all stages of oocytes examined. These results indicate that bovine oocyte maturation in culture involves modifications of the cumulus cell cytoskeleton that may regulate the onset, progression, and completion of oocyte maturation.

Differential regulation of G protein subunit expression in mouse oocytes, eggs, and early embryos.

Pertussis toxin ADP-ribosylation and Western blot analysis using G protein-specific antibodies were used to study G protein expression in mouse oocytes, eggs, and early embryos. A pertussis toxin (PT) substrate of about 40 kDa was observed in all stages, but its level was stage dependent. It decreased dramatically between germinal vesicle stage oocytes and unfertilized eggs, remained relatively constant through the early 2-cell stage, and then declined again with each cell division, reaching the lowest level at the 8- to 16-cell stage. Its level, or perhaps that of a different substrate, then increased at the blastocyst stage. Western blot analysis with antisera to the G protein alpha subunit indicated that the decrease between germinal vesicle stage oocytes and unfertilized eggs was less pronounced for the alpha subunit itself than for the PT substrate. Antisera to G protein beta subunit revealed that the difference in the amount of this subunit in germinal vesicle-stage oocytes versus unfertilized eggs was even greater than that of the PT ADP-ribosylation substrate. These results suggest that during oocyte maturation G protein beta gamma levels decline to a greater extent than alpha levels. Additional evidence supporting this hypothesis was obtained by showing that addition of exogenous beta gamma to unfertilized egg preparations increased the amount of PT substrate. These results indicate that G protein subunit expression is differentially regulated during oocyte maturation.