Alan L. Johnson

Recent studies in the hen ovary have linked the initiation of granulosa cell differentiation at follicle selection to the alleviation of inhibitory MAPK signaling. The present studies assessed a role for individual inhibitor of differentiation (Id) protein isoforms as modulators of key transcriptional events occurring within granulosa cells at or immediately subsequent to differentiation. Findings from freshly collected granulosa cells collected at different stages of follicle development demonstrated a negative association between expression levels for Id2 mRNA compared with levels of Id1, Id3, and Id4. Elevated levels of Id2 are related to a differentiating/differentiated phenotype, whereas elevated Id1, Id3, and Id4 are associated with an undifferentiated phenotype. This negative relationship extends to cell signal transduction, because factors that promote inhibitory MAPK signaling (TGF-α and betacellulin) block expression of Id2 mRNA but increase levels of Id1, Id3, and Id4. Furthermore, overexpression of Gallus Id2 in cultured granulosa was found to significantly decrease levels of Id1, Id3, and Id4 mRNA but facilitate FSHR mRNA expression and, importantly, initiate LHR mRNA expression plus LH-induced progesterone production. Finally, knockdown studies using small interfering RNA specific for Id2 revealed reduced expression of FSHR and LHR mRNA and attenuated FSH- and LH-induced levels of StAR and p450 cholesterol side-chain cleavage enzyme mRNA plus progesterone production. Collectively, these data demonstrate that Id2 expression is both sufficient and necessary for increasing LHR expression and, as a result, promoting gonadotropin-induced differentiation in hen granulosa cells subsequent to follicle selection.

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) represents one of several cytokine members of the tumor necrosis factor superfamily reported to initiate apoptosis in a wide range of transformed, but not most normal, cell types. The present studies were conducted to evaluate the potential for TRAIL to promote apoptotic cell death in differentiated granulosa cells collected from hen preovulatory follicles. While mRNA encoding critical components (including TRAIL) required for a functional extrinsic cell death pathway are expressed in granulosa cells, TRAIL treatment by itself fails to induce either caspase-3 activity or a decrease in cell viability. On the other hand, preculture of cells with the conventional chemotherapeutic, cisplatin, or the 20S proteosome inhibitor, Z-LLF-CHO, sensitizes granulosa cells to TRAIL as evidenced by enhanced caspase-3 activity after 4 h of culture and loss of cell viability after 24 h when compared with either cisplatin or Z-LLF-CHO treatment alone. Moreover, the sensitizing effect of Z-LLF-CHO on TRAIL-induced loss of cell viability is prevented by the selective caspase-8 inhibitor, Z-IETD-FMK. Interestingly, TRAIL mRNA expression is elevated both in prehierarchal follicles undergoing spontaneous atresia and in prehierarchal follicles induced to undergo atresia for 6 h in vitro. In summary, the data demonstrate the presence of a functional TRAIL signaling pathway in hen granulosa cells, and are consistent with the possibility that TRAIL signaling may directly or indirectly participate in the process of follicle atresia in vivo.

Undifferentiated granulosa cells from prehierarchal (6- to 8-mm-diameter) hen follicles express very low to undetectable levels of LH receptor (LH-R) mRNA, P450 cholesterol side chain cleavage (P450scc) enzyme activity, and steroidogenic acute regulatory (StAR) protein, and produce negligible progesterone, in vitro, following an acute (3-h) challenge with either FSH or LH. It has previously been established that culturing such cells with FSH for 18–20 h induces LH-R, P450scc, and StAR expression, which enables the initiation of progesterone production. The present studies were conducted to characterize the ability of activin and transforming growth factor (TGF) β, both alone and in combination with FSH, to promote hen granulosa cell differentiation, in vitro. A 20-h culture of prehierarchal follicle granulosa cells with activin A or transforming growth factor β (TGFβ)1 increased LH-R mRNA levels compared with control cultured cells. Activin A and TGFβ1 also promoted FSH-receptor (FSH-R) mRNA expression when combined with FSH treatment. Neither activin A nor TGFβ1 alone stimulated progesterone production after 20 h culture. However, preculture with either factor for 20 h (to induce gonadotropin receptor mRNA expression) followed by a 3-h challenge with FSH or LH potentiated StAR expression and progesterone production compared with cells challenged with gonadotropin in the absence of activin A or TGFβ1 preculture. Significantly, activation of the mitogen-activated protein (MAP) kinase pathway with transforming growth factor α (TGFα) (monitored by Erk phosphorylation) blocked TGFβ1-induced LH-R expression, and this effect was associated with the inhibition of Smad2 phosphorylation. We conclude that a primary differentiation-inducing action of activin A and TGFβ1 on hen granulosa cells from prehierarchal follicles is directed toward LH-R expression. Enhanced LH-R levels subsequently sensitize granulosa cells to LH, which in turn promotes StAR plus P450scc expression and subsequently an increase in P4 production. Significantly, the finding that TGFβ signaling is negatively regulated by MAP kinase signaling is proposed to represent a mechanism that prevents premature differentiation of granulosa cells.

As a result of searching recently available chicken (ch) expressed sequence tag databases, a new Tumor Necrosis Factor Receptor Super Family (TNFRSF) member with similarity to the murine (m) TNFRSF23 decoy receptor (DcR) has been identified. However, by comparison with the mTNFRSF23, there exist at least two splice variants of chTNFRSF23, one of which includes an intracellular death domain (TNFRSF23.v1) characteristic of death receptors, and the other with a truncated cytoplasmic domain of a DcR (named TNFRSF23.v2). These two splice variants of chTNFRSF23 display differential patterns of mRNA expression across various hen tissues, with the highest levels observed within reproductive tissues. More specifically, TNFRSF23.v1 is most highly expressed in preovulatory follicle granulosa cells in the ovary, whereas TNFRSF23.v2 mRNA is found at highest levels in ovarian stromal tissue. Primary culture experiments with granulosa cells determined that expression of TNFRSF23.v1 mRNA was decreased by protein kinase A signaling and enhanced by transforming growth factor (TGF) alpha treatment. Interestingly, TGFbeta1 and signaling via protein kinase C also enhanced levels of TNFRSF23.v1 expression but only in undifferentiated granulosa cells from prehierarchal follicles. Based on patterns of mRNA expression and its endocrine/paracrine regulation, we predict that ovarian chTNFRSF23 represents a modulator of granulosa cell survival and/or differentiation. Finally, the characterization of these receptor variants is of considerable interest from an evolutionary perspective in that they provide additional evidence to support a continuing divergence of TNFRSF members throughout vertebrate evolution.

Over the last decade, significant progress has been made towards identifying the signaling pathways within mammalian cells that lead to apoptosis mediated by death receptors. The simultaneous expression of more than one death receptor in many, if not all, cell types suggests that functional innovation has driven the divergence of these receptors and their cognate ligands. To better understand the physiological divergence of the death receptors, a phylogenetic analysis of vertebrate death receptors was conducted based upon amino-acid sequences encoding the death domain regions of currently known and newly identified members of the family. Evidence is presented to indicate an ancient radiation of death receptors that predates the emergence of vertebrates, as well as ongoing divergence of additional receptors both within several receptor lineages as well as modern taxonomic lineages. We speculate that divergence among death receptors has led to their functional specialization. For instance, some receptors appear to be primarily involved in mediating the immune response, while others play critical roles during development and tissue differentiation. The following represents an evolutionary approach towards an understanding of the complex relationship among death receptors and their proposed physiological functions in vertebrate species.

The present studies were conducted to address cellular mechanisms responsible for regulating steroidogenic acute regulatory protein (StAR) expression

Survivin is a relatively unique member of the inhibitor of apoptosis protein (IAP) family in that it contains a single baculovirus IAP repeat (BIR) domain combined with a COOH-terminal alpha-helix coiled-coil domain instead of the more common zinc-binding RING finger. Results from a variety of transformed or continuous mammalian cell lines suggest that, due to the combination of these features, Survivin is capable of regulating both cell proliferation and apoptotic cell death. However, to date there is essentially no information regarding Survivin expression, regulation or function within the ovary, or in any nonmammalian vertebrate species. In the present studies, cDNAs for chicken (ch) Survivin-142 (homologous to human Survivin-142) plus three alternatively spliced variants (ch Survivin-short, -gamma, and -delta) are described, and of these, transcripts for ch Survivin-142 and -short are expressed in granulosa cells from the hen ovary. Highest levels of Survivin mRNA during follicle development occur in mitotically active granulosa cells from undifferentiated, prehierarchal follicles. Cell cycle analysis determined that Survivin mRNA expression is elevated specifically during the G2/M phase of mitosis. Significantly, transient transfection with ch Survivin-142 in primary cultures of hen granulosa cells attenuates taxol- and N-octanoylsphingosine- (C8-ceramide-) induced caspase-3 activity, whereas overexpression of ch Survivin-short (a truncated variant that lacks much of the functional BIR domain plus the entire alpha-helix coil domain) lacks this antiapoptotic activity. Taken together, these data provide evidence for Survivin in granulosa cells acting as a bifunctional protein associated with regulation of the cell cycle and the inhibition of apoptosis.

It is now well established that vertebrate ovarian follicles undergo atresia via apoptosis, a process that is initiated within the granulosa cell lay