Observations of turkey eggs stored up to 27 days and incubated for 8 days: embryo developmental stage and weight differences and the differentiation of fertilized from unfertilized germinal discs.

For logistical reasons, egg storage prior to incubation is a growing practice in the commercial turkey industry. Yet the consequence of increasing egg storage over 7 d is a progressive increase in embryo mortality. The objective of this study was to provide the information necessary to differentiate an early dead embryo from an unfertilized egg after 8 days of incubation (DOI). Five groups of eggs each from inseminated and virgin hens were stored for progressively increasing periods of time (5-d or less, 6 to 10 d, 11 to 15 d, 16 to 20 d, and 21 to 27 d) and incubated. At 8 DOI, eggs were examined and the stage of development (Hamburger and Hamilton, 1951) and embryo weights in normally developed eggs were determined. There was a significant negative correlation between the stage of development and embryo weight with increasing storage periods. All remaining eggs from the inseminated and virgin hens were broken-out and the appearance of the yolk and the fertilized and unfertilized germinal discs examined. The yolks of both hen groups with unfertilized ova maintained a homogeneous uniform yellow-orange color. In contrast, yolks of ova that had been fertilized, with or without early-dead embryos, and yolks from virgin hens that showed evidence of parthenogenetic development (3%) had a heterogeneous appearance. Using fluorescence microscopy, the heterogeneous appearance was due to sheets of aberrant cells and less frequently dispersed cells and folds of the perivitelline layer. It was concluded that clear egg breakouts need to be performed to more accurately assess the impact of egg storage on embryonic mortality. Furthermore, such breakouts should be performed with a high intensity light directed across the surface of the germinal disc to clearly differentiate the subtle differences between an early-dead embryo and an unfertilized germinal disc.

Signaling pathways in ascidian oocyte maturation: the roles of cAMP/Epac, intracellular calcium levels, and calmodulin kinase in regulating GVBD.

Most mature ascidian oocytes undergo germinal vesicle breakdown (GVBD) when released by the ovary into sea water (SW). Acidic SW blocks this but they can be stimulated by raising the pH, increasing intracellular cAMP levels by cell permeant forms, inhibiting its breakdown or causing synthesis. Boltenia villosa oocytes undergo GVBD in response to these drugs. However, the cAMP receptor protein kinase A (PKA) does not appear to be involved, as oocytes are not affected by the kinase inhibitor H-89. Also, the PKA independent Epac agonist 8CPT-2Me-cAMP stimulates GVBD in acidic SW. GVBD is inhibited in calcium free sea water (CaFSW). The intracellular calcium chelator BAPTA-AM blocks GVBD at 10 µM. GVBD is also inhibited when the ryanodine receptors (RYR) are blocked by tetracaine or ruthenium red but not by the IP(3) inhibitor D-609. However, dimethylbenzanthracene (DMBA), a protein kinase activator, stimulates GVBD in BAPTA, tetracaine or ruthenium red blocked oocytes. The calmodulin kinase inhibitor KN-93 blocks GVBD at 10 µM. This and preceding papers support the hypothesis that the maturation inducing substance (MIS) produced by the follicle cells in response to increased pH causes activation of a G protein which triggers cAMP synthesis. The cAMP then activates an Epac molecule, which causes an increase in intracellular calcium from the endoplasmic reticulum ryanodine receptor. The increased intracellular calcium subsequently activates calmodulin kinase, which causes an increase in cdc25 phosphatase activity, activating MPF and the progression of the oocyte into meiosis.

Identification of germinal disk region derived genes potentially involved in hen fertility.

Despite the regular decrease in fertility observed in hens, especially in "meat" lines, little is known about genes affecting fertility. We used the Affymetrix microarray to search for oocyte genes whose expression would vary in relation to fertility rate in both "laying" and "meat" line hens. We focused on oocyte genes because several of them have been found to be involved in fertility in other species. Based on microarray analysis, 54 and 84 genes were differentially expressed between germinal disc regions (GDR) of F1 maturation stage oocytes from hens exhibiting either high (100%) or low (from 22% to 80%) fertility rate from laying and meat lines respectively. Most of these differentially expressed genes were distributed between "laying" and "meat" lines indicating that mechanisms involved in the decrease in fertility rates in these two cases were independent. Real time RT-PCR performed on the same samples which were used for microarray confirmed in several cases differences in gene expression levels detected by microarray. Moreover the correlations between gene expression levels and fertility rates were evaluated for the 10 most interesting genes at different stages of follicular maturation and early embryo development on individual GDR samples from hens exhibiting different fertility rates. In total, we identified five genes whose expression levels correlated with fertility rate in accordance with findings of microarray analysis and real time RT-PCR: VWC2, CR407412, TAPA, FGL2, and TRAP6. The biological significance of these genes sheds light on potential mechanisms influencing fertility and could provide candidates for fertility markers in the hen.

Oocyte maturation: converting the zebrafish oocyte to the fertilizable egg.

The process of oogenesis culminates in steroid-induced oocyte maturation to produce the fertilizable egg. A quintessential biological entity, the egg is central to the production of new individuals. The result of egg fertilization by a sperm cell is the production of the mother of all stem cells (i.e. the zygote). Furthermore, the egg cytoplasm is the only one known to support reprogramming a transplanted nucleus to give rise to an individual (i.e. animal cloning). Zebrafish oocyte maturation is a complex event encompassing a number of cellular changes including germinal vesicle migration (GVM) and dissolution or breakdown (GVD), ooplasmic clearing (OC) with correlated yolk protein changes (YP), development of osmoregulation (OR) in fresh water, the formation of the future embryonic pole, the blastodisc (BF) and activatibility (AC) or cortical maturation. In zebrafish, and many other teleosts, 17alpha, 20beta-dihydroxy-4-pregnen-3-one (17alpha, 20beta-DP) has been shown to be the normal inducer of oocyte maturation. A 17alpha, 20beta-DP membrane-resident receptor mediates oocyte maturation via non-genomic mechanisms that are beginning to be understood. This paper will highlight some of the cellular markers resulting from the signaling initiated by 17alpha, 20beta-DP. By describing these markers, it is hoped that workers in the field will have additional tools to help further elucidate the signaling events of oocyte maturation.