The detailed analysis of the lipid composition of immature pig oocytes represents the first such study carried out on mammalian eggs. In order to undertake a large scale lipid analysis using conventional extraction and chromatographic techniques a procedure for mass harvesting relatively large numbers of pig oocytes (200\p=n-\300 oocytes/ovary) was developed. The study revealed that triacylglycerol was the major lipid component (100\m=.\71nmol/mg protein) followed by cholesterol (32\m=.\71nmol/mg protein). Phosphatidylcholine constituted the major phospholipid component (27\m=.\83nmol/mg protein). Pig oocytes contained relatively low proportions of phosphatidylethanolamine (16\m=.\41% total phospholipid) and relatively high proportions of lysophosphatidylcholine (4\m=.\68% total phospholipid). The free fatty acid pattern was strikingly similar to the fatty acid composition of phosphatidylcholine. This observation, in conjunction with the observed high levels of lysophosphatidylcholine and the low ratio of phosphatidylethanolamine to phosphatidylcholine, suggests a fast rate of phospholipid turnover in the immature pig oocyte. Analysis of fatty acids esterified to the individual phospholipids and neutral lipids has shown that in all the classes examined, particularly in the neutral lipid fractions, there are high levels of the saturated fatty acid palmitic acid (16:0) and the monounsaturated fatty acid oleic acid (18:1). Triacylglycerol, free fatty acids and most of the phospholipids, particularly phosphatidylethanolamine, are considerably enriched in n-6 polyunsaturated fatty acids, specifically linoleic (18:2), arachidonic (20:4) and adrenic (22:4) acids. This may indicate an ability of oocytes to synthesize prostaglandins and leukotrienes.The results show that the lipid environment of the immature pig oocyte may be adapted to the highly specialized requirements of the cell, promoting growth and development with a potential role in the regulation of maturation.

Folliculogenesis is the process of development of ovarian follicles that ultimately results in the release of fertilizable oocytes at ovulation. This is a complex program that involves the proliferation and differentiation of granulosa cells. Granulosa cells are necessary for follicle growth and support the oocyte during folliculogenesis. Genes that regulate the proliferation and differentiation of granulosa cells are beginning to be elucidated. In this study, the expression patterns of Notch receptor genes and their ligands, which have been shown to regulate cell-fate decisions in many systems during development, were examined in the mammalian ovary. In situ hybridization data showed that Notch2, Notch3, and Jagged2 were expressed in an overlapping pattern in the granulosa cells of developing follicles. Jagged1 was expressed in oocytes exclusively. Downstream target genes of Notch also were expressed in granulosa cells. These data implicate the Notch signaling pathway in the regulation of mammalian folliculogenesis.

Pig oocytes, with (intact) or without (denuded) adhering granulosa cells, were cultured for 33 to 35 h in minimal culture media containing bovine serum albumin and either progesterone or estradiol-17beta or both steroids. The incidence of maturation for intact oocytes in minimal medium was comparable, if not superior, to that observed by other investigators using complex culture media. No statistically significant stimulation of maturation by progesterone was observed for either intact or denuded oocytes. Estradiol-17beta at concentrations of 1.0 and 10.0 mug per ml inhibited the maturation of denuded oocytes, but the inhibition was overcome either by including progesterone in the medium or by subsequently culturing the inhibited oocytes in steriod-free medium. Chromosomal examination of air-dried oocytes indicated that intact and denuded oocytes cultured either in progesterone or estradiol-17beta exhibited an increased incidence of diploidy and hyperhaploidy at telophase I and metaphase II, but that oocytes cultured in media containing both progesterone and estradiol showed a higher incidence of normal haploidy than did control oocytes.

The cytogenetic configurations of germinal vesicle (gv) chromatin were analyzed for pools of porcine oocytes harvested from small (1.0-2.0 mm), medium (3.0-5.0 mm), and large (6.0-10.0 mm) antral follicles. Groups of oocytes from these follicular classes also were examined by high-resolution, two-dimensional, polyacrylamide gel electrophoresis to compare their patterns of polypeptide synthesis. The results show a high incidence of gross and cytogenetic degeneration among oocytes from small antral follicles as compared with those from medium or lage follicles. Pools of oocytes could be separated, on the basis of gross morphology and integrity of adherent granulosa cells, into two classes: "Type A" which appeared normal, and "type B" which appeared to be atretic. Among selected "type A" oocytes a particular chromatin configuration, termed "fibrous" characterizes the gv of oocytes from small follicles; whereas a different configuration, termed "diffuse," characterizes the gv of oocytes from large follicles. The patterns of polypeptide synthesis were markedly different for samples of "type A" oocytes of the three follicular classes; and the patterns for oocytes from medium and large follicles were more similar to each other than to patterns for oocytes from slall follicles. The incidences of maturational development beyond the gv stage in vitro were similar for "type A" oocytes from the three follicular classes (i.e., 66% to 82% maturation); although "type B" oocytes underwent maturation beyond the gv at a significantly reduced incidence (i.e., 20% to 29% maturation). "Type A" oocytes from large follicles completed maturation in vitro (i.e., underwent the first meiotic division) at a significantly higher incidence (55%) than did oocytes from small (11% to 20%) or medium (16%) follicles. Our findings are consistent with the hypotheses that a high proportion of oocytes from small antral follicles are atretic, and that a developmental program controls the molecular and cytogenetic changes occurring in porcine oocytes during follicular growth. These changes appear to be highly correlated with the acquisition of competency to complete maturation in vitro, and possibly also are required for normal fertilization and embryogenesis.

Mammalian eggs and embryos possess a major cytoskeletal network composed of large planar "sheets" distributed throughout the cytoplasm. Cytoskeletal sheets are found neither in mammalian somatic cells nor in eggs or embryos of non-mammals. In this study, we have investigated the structural composition of the sheets in eggs and embryos of the golden Syrian hamster by (1) analysis of replicas from quick-frozen, deep-etched specimens, (2) analysis of thick, resin-embedded specimens using an intermediate voltage electron microscope (IVEM), (3) laser diffraction of EM images, (4) differential extraction with detergents, and (5) immunocytochemistry. Our results indicate that each sheet is composed of two closely apposed arrays of 10-nm filaments. Each filament within an array is held in register with its neighbor by lateral cross-bridges and the two parallel arrays of filaments are interconnected by periodic cross-bridges about 20 nm in length. Laser diffraction of negatives from IVEM images indicates that each array is composed of fibers that form a square lattice, and the two arrays are positioned in register by cross-bridges forming a single sheet. This lattice forms the skeleton of the sheets which is covered with a tightly packed layer of particulate material. By incubation in media containing different ratios of mixed-micelle detergents, it is possible to remove components sequentially from the sheets and to extract the particulate material. Immunocytochemical localization demonstrates that the sheets bind antibodies to keratin, and to a small extent actin, but do not bind antibodies to vimentin or tubulin. Examination of sheets within embryos at the time of embryonic compaction demonstrates that the sheets begin to fragment and disassemble in regions of blastomeres where desmosomes form, but undergo no structural alterations in interior and basal surfaces of the blastomeres. In regions of blastomere-blastomere contact the sheets fragment and associate with granules resembling keratohyalin granules found in keratinocytes.

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Fertilization of the mammalian egg initiates numerous biochemical and structural changes which remodel the egg into a single-celled zygote. To date, the most extensively studied phenomenon of fertilization in virtually all species has been the relationship between sperm penetration and the induction of the initial rise in intracellular-free calcium ([Ca2+]i) concentration within the egg. In contrast, relatively few studies have focused on the biochemical events following this rise in calcium, and even fewer studies have directly linked the biochemical events to the structural changes which must ensue for proper development of the embryo. In this study, we exploited recently developed technologies to investigate the action of protein kinase C (PKC), a presumed downstream transducer of the initial rise in [Ca2+]i, during fertilization and artificial activation with calcium ionophore or phorbol 12-myristate 13-acetate (PMA). The newly developed myristoylated PKC pseudosubstrate (myrPKC psi) was used to specifically inhibit PKC, thereby averting the trauma of injecting the egg with nonmyristoylated PKC psi. Following fertilization, eggs which were pretreated with myr-PKC psi were not capable of forming a second polar body and pronuclear formation was significantly inhibited. Spatial and temporal localization of PKC using confocal microscopy to visualize the PKC reporter dye, Rim-1, demonstrated localization of PKC to the lateral aspects of the forming second polar body after fertilization, or after artificial activation with calcium ionophore or PMA. In vivo biochemical analysis of eggs which were fertilized or artificially activated demonstrated that PKC activity rose at the same time (40 min) as the second polar body formed and then subsided over the next 5 hr post activation. From these data, we conclude that PKC plays an integral role in directing the transformation from egg to embryo.

Several isotypes of protein kinase C (PKC) have been reported to be expressed in mammalian eggs, but it is unknown whether these isotypes have a common function in the egg during or within the first few hours of fertilization. Here we show that the isotypes of PKC exhibit distinct patterns of enrichment immediately after mouse egg activation. PKCalpha and gamma accumulate in the egg cortex 25 min post-activation, while only PKCalpha accumulates at the contractile ring of the forming second polar body about 1.5 h post-activation. PKCzeta exhibits some unique features that resulted in it being the focus of more extensive analysis. PKCzeta is tightly associated with the meiotic spindle as determined by detergent extraction and is closely associated with alpha-tubulin as determined by FRET analysis in the metaphase II (MII) egg. In addition, after egg activation, PKCzeta remains associated with the spindle as it transits into anaphase II and later telophase II, becoming associated with the midzone microtubules. Antibodies to the active form of PKCzeta are enriched on the spindle poles and later in development on the midzone microtubules. Active PKCzeta also is enriched in both pronuclei in the 6-h post-fertilization and in the 14-h post-fertilization embryo as well as in the nuclei of the two-cell embryo. Inhibition of PKCzeta, but not inhibition of other isotypes of PKC, results in rapid disruption of the meiotic spindle. This study suggests that PKCzeta has a role in spindle stability, while other PKC isotypes have different roles in the conversion of the egg to the zygote.