Early embryonic development in many organisms relies upon maternal molecules deposited into the egg prior to fertilization. We have cloned and characterized a maternal T-box gene in the zebrafish, eomesodermin(eomes). During oogenesis, the eomes transcript becomes localized to the cortex of the oocyte. After fertilization during early cleavage stages, eomes is expressed in a vegetal to animal gradient in the embryo, whereas Eomesodermin protein (Eom) is distributed cytoplasmically throughout the blastoderm. Strikingly, following midblastula transition, nuclear-localized Eomesodermin is detected on the dorsal side of the embryo only. Overexpression of eomes results in Nodal-dependent and nieuwkoid/dharma (nwk/dhm) independent ectopic expression of the organizer markers goosecoid (gsc), chordin (chd) and floating head (flh) and in the formation of secondary axes. The same phenotypes are observed when a VP16-activator construct is injected into early embryos, indicating that eomes acts as a transcriptional activator. In addition, a dominant-negative construct and antisense morpholino oligonucleotides led to a reduction in gsc and flh expression. Together these data indicate that eomes plays a role in specifying the organizer.

The Fas antigen is a cell surface receptor that triggers apoptosis when bound to Fas ligand (FasL). Studies were undertaken to determine whether the cow provides a suitable model to study the role of the Fas pathway in inducing apoptosis of ovarian cells during follicular atresia. Expression of Fas antigen mRNA and responsiveness to FasL-induced killing in vitro were measured. Effects of the cytokines tumor necrosis factor (TNF)-alpha and interferon-gamma (IFN) were studied because of previous demonstrations of their role in Fas-mediated apoptosis in other cell types. Fas antigen mRNA was detectable in cultured granulosa and theca cells, and expression was increased by treatment with IFN but not TNF. Granulosa and theca cells were resistant to FasL-induced killing unless pretreated with IFN. TNF had no effect on FasL-induced killing. Granulosa and theca cell cultures in which killing occurred in response to FasL stained positively for annexin V, an early marker for cells undergoing apoptosis. These results provide a basis for further studies using the bovine ovary to examine the role of the Fas antigen in follicular atresia.

Fas antigen is a receptor that triggers apoptosis when bound by Fas ligand (FasL). A role for Fas antigen in follicular atresia was studied in follicles obtained during the first wave of follicular development during the bovine estrous cycle (estrus is Day 0). Granulosa and theca cells were isolated from healthy dominant follicles and the two largest atretic subordinate follicles on Day 5, atretic dominant follicles on Days 10-12, and preovulatory follicles on Day 1. Fas antigen mRNA levels were highest in granulosa cells from subordinate as compared to other follicles, and lowest in theca cells from healthy Day 5 dominant as compared to other follicles. FasL alone had no effect on viability of granulosa or theca cells but became cytotoxic in the presence of interferon-gamma (IFN). IFN has been shown to induce responsiveness to Fas antigen-mediated apoptosis in other cell types. In the presence of IFN, killing of granulosa cells by FasL was greater in subordinate compared to healthy dominant follicles on Day 5, did not differ between healthy and atretic dominant follicles, and was similar in theca among all follicles. Granulosa cells from preovulatory follicles, which had been exposed to the LH surge in vivo, were completely resistant to FasL-induced killing. In summary, Fas antigen expression, and responsiveness to Fas antigen-mediated apoptosis, vary during follicular development.