Cellular mechanisms and modulation of activin A- and transforming growth factor beta-mediated differentiation in cultured hen granulosa cells.

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) beta, 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 beta (TGFbeta)1 increased LH-R mRNA levels compared with control cultured cells. Activin A and TGFbeta1 also promoted FSH-receptor (FSH-R) mRNA expression when combined with FSH treatment. Neither activin A nor TGFbeta1 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 TGFbeta1 preculture. Significantly, activation of the mitogen-activated protein (MAP) kinase pathway with transforming growth factor alpha (TGFalpha) (monitored by Erk phosphorylation) blocked TGFbeta1-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 TGFbeta1 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 TGFbeta signaling is negatively regulated by MAP kinase signaling is proposed to represent a mechanism that prevents premature differentiation of granulosa cells.

Alternatively spliced variants of Gallus gallus TNFRSF23 are expressed in the ovary and differentially regulated by cell signaling pathways.

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.

All in the family: evolutionary and functional relationships among death receptors.

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.

Relationship between steroidogenic acute regulatory protein expression and progesterone production in hen granulosa cells during follicle development.

The present studies were conducted to address cellular mechanisms responsible for regulating steroidogenic acute regulatory protein (StAR) expression and progesterone synthesis at maturational stages corresponding to both the time of hen follicle selection, as well as before and after the LH surge in preovulatory follicle granulosa cells. A recently published report has established that mitogen-activated protein (MAP) kinase signaling induced by transforming growth factor alpha (TGFalpha) treatment blocks FSH-induced differentiation and StAR expression in cultured hen granulosa cells, whereas inhibitors of MAP kinase signaling enhance FSH-induced differentiation. The present in vitro studies demonstrate that in addition to MAP kinase signaling, activation of protein kinase C (PKC) blocks both FSH-induced StAR expression and the initiation of progesterone production in prehierarchal follicle granulosa cells, whereas the pharmacologic inhibitor of PKC, GF109203X, potentiates FSH-induced StAR expression and, as a consequence, the initiation of progesterone synthesis. Moreover, we demonstrate in granulosa cells collected from preovulatory follicles that although an acute increase in progesterone production in response to LH treatment requires rapid transcription and translation of StAR, the magnitude of progesterone production is rate-limited by one or more factors other than StAR (e.g., the P450 cholesterol side-chain enzyme). Finally, the rapid turnover of StAR protein, such as occurs following the withdrawal of LH, provides an additional mechanism for the tight regulation of progesterone production that occurs during the hen ovulatory cycle, and explains the rapid loss of steroidogenesis in the postovulatory follicle. In summary, data reported herein support the proposal that paracrine/autocrine factors (including but not necessarily limited to TGFalpha) prevent premature expression of StAR in prehierarchal follicle granulosa cells by more than one receptor-mediated signaling pathway. Furthermore, subsequent to follicle selection into the preovulatory hierarchy, StAR transcription and translation is necessary but not sufficient for the full potentiation of the preovulatory surge of serum progesterone.

Survivin as a cell cycle-related and antiapoptotic protein in granulosa cells.

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.

Expression and regulation of Fas antigen and tumor necrosis factor receptor type I in hen granulosa cells.

It is now well established that vertebrate ovarian follicles undergo atresia via apoptosis, a process that is initiated within the granulosa cell layer of undifferentiated follicles. Although the exact signals, membrane-bound receptors, and associated intracellular signaling pathways leading to apoptosis within granulosa cells have yet to be established, it is evident that multiple and redundant pathways exist. Fas, together with its ligand, has been the most commonly studied death-inducer in the mammalian ovary; however, nothing is currently known regarding expression of either Fas or the related tumor necrosis factor receptor type 1 (TNFR1), in avian species. Based on characterization of a chicken fas partial cDNA, which includes the entire death domain, the deduced amino acid sequence shows 37% identity (53% positive) to human Fas. Northern blot analysis demonstrates low expression of the 2.0-kilobase fas transcript in most tissues, including the granulosa layer, and highest levels are found in the spleen, theca tissue, and the postovulatory follicle. Significantly, fas and tnfr1 mRNA levels are higher in atretic follicles than in nonatretic, prehierarchal (3- to 8-mm diameter) follicles. Moreover, both fas and tnfr1 mRNA levels are up-regulated by twofold to eightfold in granulosa cells following plating in the presence of fetal bovine serum, with the most dramatic increase found in fas expression within prehierarchal follicle granulosa. Coculture with transforming growth factor (TGF) beta attenuates this increase for both receptors, whereas cAMP attenuates only the up-regulation of fas. By comparison, treatment with TGFalpha enhances expression of tnfr1, but not fas, mRNA. Taken together, these data are the first to implicate fas as a mediator of granulosa cell apoptosis in a nonmammalian vertebrate, and to implicate the protein kinase A signaling pathway in down-regulating fas expression. In addition, data provided demonstrate the presence of multiple death domain-containing TNFR family members simultaneously expressed within hen granulosa cells, each of which may be regulated by separate signaling pathways.

Conservation of death receptor-6 in avian and piscine vertebrates.

One of the most recently identified members of the tumor necrosis factor receptor family, death receptor-6 (DR6), has been shown to mediate apoptosis following overexpression in HeLa cells. The avian and piscine orthologs of DR6 have now been identified, and the deduced amino acid sequence for each demonstrates a high level of conservation compared to the mammalian sequence. Expression of dr6 mRNA occurs widely across tissues of both the mature chicken and brook trout. It is now well-established that ovarian follicular atresia occurs via apoptosis originating within the granulosa cell layer. Accordingly, DR6 expression within the ovary was examined to assess the relationship between stage of follicle development and relative levels of this death receptor. Of particular interest was the finding that elevated levels of dr6 mRNA, as well as the translated protein, are expressed in atretic compared to healthy follicles of the hen ovary, thus providing the first association between DR6 expression and apoptosis, in vivo. We conclude that DR6 is a highly conserved and widely expressed death-domain-containing receptor and may be implicated in regulating follicle atresia within the vertebrate ovary.

Regulation of steroidogenic acute regulatory protein and luteinizing hormone receptor messenger ribonucleic acid in hen granulosa cells.

The regulation of steroidogenic acute regulatory protein (StAR) in vitro by gonadotropins was investigated in granulosa cells from prehierarchal and preovulatory hen follicles. Basal levels of StAR messenger RNA (mRNA) in undifferentiated granulosa cells from prehierarchal (6- to 8-mm) follicles were consistently low, but detectable, and were significantly increased by treatment with 8-bromo-cAMP and FSH (but not LH) within 3-6 h of culture. After 20 h of culture, 8-bromo-cAMP, FSH, and LH each increased StAR mRNA levels above those in control cultured cells, and the delayed response to LH treatment was associated with increased levels of LH receptor (LH-R) mRNA. On the other hand, inhibition of mitogen-activated protein (MAP) kinase signaling, using the MAP kinase kinase inhibitors U0126 and PD98059, in the presence of FSH further increased StAR mRNA and protein levels, LH-R mRNA levels, and progesterone synthesis compared with those in cells cultured with FSH alone. The highest basal expression of StAR mRNA during follicle development was found in granulosa from the largest (F1) preovulatory follicle, with comparatively lower levels in granulosa from less mature (F2 plus F3) preovulatory follicles. Treatment with LH rapidly increased StAR mRNA and protein (but not LH-R mRNA) expression in cultures of F1 granulosa and in combined F2 plus F3 granulosa within 3 h, although the magnitude of stimulation was greater in F2 plus F3 granulosa. Compared with results from granulosa cells from prehierarchal follicles cultured for 20 h, inhibition of MAP kinase signaling in the presence of LH for 1 h failed to further enhance levels of StAR or LH-R expression or progesterone production in F2 plus F3 follicle granulosa compared with the effect of LH treatment alone. These results demonstrate that StAR expression in the hen ovary is up-regulated by gonadotropins at least in part via cAMP signaling. The ability of MAP kinase kinase inhibitors to potentiate gonadotropin-induced StAR and LH-R expression plus progesterone synthesis in prehierarchal follicle granulosa cells in vitro suggests that inhibition of paracrine or autocrine factor-mediated MAP kinase signaling in vivo may be a prerequisite for the full potentiation of granulosa cell steroidogenesis that occurs after recruitment into the preovulatory hierarchy. Finally, these results fail to support a role for MAP kinase signaling in acutely modulating LH-mediated StAR expression or progesterone production in hierarchal follicles, such as occurs during the preovulatory surge of progesterone.

Activation of the Akt/protein kinase B signaling pathway is associated with granulosa cell survival.

Follicles from the hen ovary that have been selected into the preovulatory hierarchy are committed to ovulation and rarely become atretic under normal physiological conditions. In part, this is attributed to the resistance of the granulosa layer to apoptosis. The present studies were conducted to evaluate the role of the phosphatidylinositol (PI) 3-kinase/Akt signaling pathway in hen granulosa cell survival and, by implication, follicle viability. Cloning of the chicken akt2 homologue revealed a high degree of amino acid homology to its mammalian counterparts within the catalytic domain, plus complete conservation of the putative Thr(308) and Ser(474) phosphorylation sites. Treatment of granulosa cells from the three largest preovulatory follicles with insulin-like growth factor (IGF)-I and, to a lesser extent, transforming growth factor (TGF)-alpha induces rapid phosphorylation of Akt, and such phosphorylation is effectively blocked by the PI 3-kinase-inhibitor LY294006. Serum withdrawal from cultured cells for 33-44 h initiates oligonucleosome formation, an indicator of apoptotic cell death, whereas cotreatment with IGF-I prevents this effect. Moreover, treatment of cultured cells for 20 h with LY294006 induces apoptosis. The potential for nonspecific cell toxicity following LY294006 treatment is considered unlikely because of the ability of either LH or 8-bromo cAMP cotreatment to block LY294006-induced cell death. Finally, both IGF-I and TGF-alpha also activate mitogen-activated protein (MAP) kinase signaling, at least in part, through the phosphorylation of ERK: However, treatment with neither U0126 nor PD98059 (inhibitors of MAP kinase kinase) induced cell death in cultured granulosa cells, despite the ability of each inhibitor to effectively block Erk phosphorylation. Taken together, these results provide evidence for a role of the Akt signaling pathway in promoting cell survival within the preovulatory follicle granulosa layer. In addition, the data indicate the importance of an alternative survival pathway mediated via gonadotropins and protein kinase A independent of Akt signaling.

Conservation of steroidogenic acute regulatory (StAR) protein structure and expression in vertebrates.

Complementary DNAs for the open reading frames of the chicken, Xenopus and zebrafish StAR homologs were cloned along with a partial cDNA of the zebrafish homolog to MLN64, a StAR-related protein. A comparison of the amino acid sequences of piscine, amphibian, avian and mammalian StARs, indicates strong conservation of the protein across divergent vertebrate groups. On Northern blots probed with species specific StAR cDNAs, expression of StAR transcripts was observed in the ovary and adrenal of chicken, and the ovary, testis, kidney and head of zebrafish. The expression of StAR mRNA in various compartments of the hen ovary was consistent with the results of past studies on steroidogenesis; expression was first observed in follicles selected into the preovulatory hierarchy and was greatest in the largest preovulatory follicle. The expression of StAR mRNA was also consistent with aromatase expression in zebrafish ovaries. The conserved deduced protein sequence and expression pattern of StAR transcripts in chicken and zebrafish tissues, strongly suggest that StAR is also involved in the regulation of steroidogenesis in nonmammalian vertebrates.

Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays.

We describe a novel sequencing approach that combines non-gel-based signature sequencing with in vitro cloning of millions of templates on separate 5 microm diameter microbeads. After constructing a microbead library of DNA templates by in vitro cloning, we assembled a planar array of a million template-containing microbeads in a flow cell at a density greater than 3x10(6) microbeads/cm2. Sequences of the free ends of the cloned templates on each microbead were then simultaneously analyzed using a fluorescence-based signature sequencing method that does not require DNA fragment separation. Signature sequences of 16-20 bases were obtained by repeated cycles of enzymatic cleavage with a type IIs restriction endonuclease, adaptor ligation, and sequence interrogation by encoded hybridization probes. The approach was validated by sequencing over 269,000 signatures from two cDNA libraries constructed from a fully sequenced strain of Saccharomyces cerevisiae, and by measuring gene expression levels in the human cell line THP-1. The approach provides an unprecedented depth of analysis permitting application of powerful statistical techniques for discovery of functional relationships among genes, whether known or unknown beforehand, or whether expressed at high or very low levels.

Caspase-3 and -6 expression and enzyme activity in hen granulosa cells.

We have cloned and sequenced cDNAs corresponding to the complete coding regions of the chicken homologues to mammalian caspase-3 and caspase-6. Both caspases are included among members of the cysteine protease (caspase) family that are most closely identified with mediating apoptosis. The deduced amino acid sequences for chicken caspase-3 and -6 show 65% and 68% identity with the respective human sequences, with complete conservation found within the QACRG active peptide region. Both caspase-3 and -6 are widely expressed within various tissues from the hen. Within the ovary, levels of caspase-3 and caspase-6 mRNA and protein do not change significantly in theca tissue during follicle development. On the other hand, procaspase-3 and -6 protein levels are elevated by 2- to 5-fold in preovulatory, compared to prehierarchal (6- to 8-mm diameter), follicle granulosa cells. Nevertheless, the function of this family of cell death-inducing proteins requires activation of the proenzyme caspase, which occurs after cleavage at predictable sites within the N-terminal domain. Accordingly, it was determined that okadaic acid, a pharmacologic inducer of apoptotic cell death in cultured apoptosis-resistant, preovulatory follicle granulosa cells, induced both caspase-3- and caspase-6-like activity within 8-16 h of treatment. By comparison, spontaneous apoptotic cell death that occurs in apoptosis-sensitive, prehierarchal follicle granulosa cells after short-term suspension culture is accompanied by a more rapid increase (within 2 h) in both caspase-3- and -6-like activity. Treatment with 8-bromo-cAMP, which has previously been shown to attenuate, or at least slow, the onset of apoptosis in prehierarchal follicle granulosa cells, mitigates this suspension culture-induced increase in caspase activity. While the present results provide further support for the relationship between caspase activation and apoptotic cell death in hen granulosa cells, the molecular ordering of enzymatic events and the caspase-specific substrates remain to be elucidated.

Bcl-X(LONG) protein expression and phosphorylation in granulosa cells.

Expression of Bcl-x protein was evaluated in hen ovarian follicles, relative to stage of development, and in cultured granulosa cells after treatment with various apoptosis-suppressing or -inducing agents. Using Western blot analysis, Bcl-X(LONG) was most frequently observed migrating as a doublet with a molecular mass of approximately 28 kDa; the apparent higher molecular mass band of this doublet was determined to represent a phosphorylated form. Consistent with previous findings reported for bcl-x messenger RNA, only the Bcl-X(LONG) (apoptosis-suppressing) form of protein was detected in the hen granulosa cells, and highest levels of Bcl-X(LONG) protein (sum of the protein doublet) expression were found in granulosa from preovulatory follicles together with tissues with immune function (e.g. spleen and bone marrow). Evidence for Bcl-X(SHORT) expression was found only in the theca and several nonovarian tissues. Immunocytochemical analysis of preovulatory vs. prehierarchal follicles confirmed the comparatively greater expression of cytoplasmic Bcl-X(LONG), particularly in preovulatory follicle granulosa. Levels of Bcl-X(LONG) were significantly increased after 20 h of culture in the presence of 8-bromo-cAMP (8-br-cAMP; compared with culture in control medium) in granulosa cells from both stages of follicle development. Such results are correlated with the protein's proposed function to protect against cell death in apoptosis-resistant, preovulatory follicle granulosa cells and are consistent with the ability of this cAMP agonist to increase bcl-X(LONG) messenger RNA levels in cultured cells. Furthermore, several factors that have previously been demonstrated to suppress apoptosis in granulosa cells, in vitro, (e.g. 8-br-cAMP, LH, FSH) were found to rapidly (within 15 min) increase levels of phosphorylated Bcl-X(LONG), compared with control cells, whereas an inhibitor of protein kinase A (H-89) blocked such phosphorylation. By comparison, transforming growth factor alpha, a factor previously found to attenuate apoptosis and apoptosis-inducing agents (e.g. paclitaxel, C8-ceramide, daunorubicin, UV irradiation) failed to phosphorylate Bcl-X(LONG). From these studies, it is concluded that both the phosphorylation of Bcl-X(LONG) (a short-term response) and increased levels of Bcl-X(LONG) (a comparatively slower response) in hen granulosa cells are promoted by gonadotropins via the adenylyl cyclase/cAMP signaling pathway. Moreover, elevated levels of chicken Bcl-X(LONG) protein expression and its phosphorylated state are correlated with resistance to apoptotic cell death in granulosa cells in vitro and ultimately a resistance to ovarian follicle atresia in vivo.

Characterization of the chicken interleukin-1beta converting enzyme (caspase-1) cDNA and expression of caspase-1 mRNA in the hen.

We have cloned and sequenced a chicken homolog to the mammalian interleukin-1beta (IL-1beta)-converting enzyme (caspase-1) cDNA and have evaluated caspase-1 mRNA expression in various tissues from the domestic hen, including ovarian follicle granulosa and theca layers. The deduced amino acid (aa) sequence of chicken caspase-1 is 44.9% identical to human caspase-1, and contains an active site pentapeptide that is characteristic of the caspase family of cysteine proteases. Of interest, however, is that the putative chicken caspase-1 cDNA is predicted to encode a comparatively short (19aa) N-terminal prodomain, as well as two Cys residues within the active pentapeptide (QC162C163RG) as compared to the QACRG pentapeptide found in the mammalian caspase-1 sequence. While the chicken caspase-1 mRNA transcript is widely expressed among different tissues, levels are particularly high in the bursa of Fabricius and comparatively low in ovarian follicles at all stages of development. Finally, treatment of granulosa cells with IL-1beta, the primary if not sole product of caspase-1 activity, fails to either promote apoptotic cell death or enhance viability in granulosa cells. Considering the relatively low levels of caspase-1 mRNA expression in ovarian follicle tissues plus the inability of IL-1beta to alter granulosa cell viability, in vitro, it is concluded that caspase-1 is not an integral part of the apoptotic pathway in granulosa cells. However, the pattern of mRNA expression is consistent with a requirement for caspase-1 mediated IL-1beta production in chicken immune tissues.

Expression of the inhibitor of T-cell apoptosis (ita) gene in hen ovarian follicles during development.

It is now well established that within the hen ovary, preovulatory follicles rarely become atretic and that granulosa cells from preovulatory follicles are relatively resistant to undergoing apoptosis in vitro. By comparison, prehierarchal (< or = 8-mm diameter) follicles are highly susceptible to becoming atretic in vivo, and approximately 70% of granulosa cells collected from 3- to 8-mm-diameter follicles rapidly undergo apoptosis when incubated for as little as 6 h in vitro in defined medium. The present studies were conducted to characterize expression of an inhibitor of apoptosis (iap) gene, inhibitor of T-cell apoptosis (ita), within hen follicle tissues at various stages of follicle development. The ita gene product has recently been shown to share homology within both the baculovirus repeat sequences of the N-terminus and the zinc ring-finger motif from the C-terminus and was originally determined to be expressed in chicken cells of T-lymphocyte lineage. In the present studies, highest levels of ita mRNA within the granulosa cell layer were found in preovulatory (atresia-resistant) follicles, with significantly lower levels detected in prehierarchal follicles. After 24 h of primary culture, ita mRNA levels increased in granulosa cells from preovulatory follicles by 3.2-fold as compared to those in freshly collected cells and were elevated by 8.9-fold in those granulosa cells from 6- to 8-mm follicles that successfully formed a primary culture monolayer. Moreover, ita mRNA levels were significantly increased in 6- to 8-mm-follicle granulosa cells after only 2 h of suspension culture, and this increase could be prevented by actinomycin D. This spontaneous increase in ita expression may serve to protect from cell death the relatively small population of prehierarchal follicle granulosa cells that survive in vitro. It is concluded from these data, taken together, that patterns of ita mRNA expression during follicle development are consistent with a potential role for this gene in protecting granulosa cells from apoptosis and thus maintaining follicle viability.

Expression of bcl-2 and nr-13 in hen ovarian follicles during development.

Follicle atresia is initiated within the granulosa cell layer of ovarian follicles and is mediated via the process of apoptosis. In the hen, at least two populations of granulosa cells can be distinguished during follicle development on the basis of their inherent susceptibility or resistance to apoptosis, in vitro. Given the previously established correlation between expression of bcl-xLong and hen granulosa cell resistance to apoptosis, the present studies were conducted to characterize expression of bcl-2 and an avian bcl-2 homologue, nr-13, in follicles at various stages of development. Levels of nr-13 mRNA were significantly higher only in granulosa cells from the largest (F1) preovulatory follicle compared to 3- to 5-mm prehierarchal follicles. By comparison, bcl-2 mRNA levels were 5- to 9-fold higher in granulosa cells from the three largest preovulatory follicles compared to those from follicles 9 to 12 mm in diameter and prehierarchal follicles. The increase in neither nr-13 nor bcl-2 was correlated with the stage of follicle development associated with the acquisition of resistance to apoptosis in granulosa cells (e.g., at the 9- to 12-mm stage). Results from the present studies do not support a close correlation between constitutive expression of nr-13 or bcl-2 mRNA and the transition from a state of apoptosis susceptibility to apoptosis resistance in hen granulosa cells. Thus, it is proposed that nr-13 and bcl-2 play more of a supportive role in regulating additional aspects of ovarian cell function such as cell proliferation and/or differentiation.

Characterization of the avian Ich-1 cDNA and expression of Ich-1L mRNA in the hen ovary.

We have sequenced the chicken interleukin-1beta-converting enzyme (ICE) and ced-3 homolog (Ich-1) cDNA, and evaluated Ich-1 mRNA expression in the hen ovary during follicle development. While two alternatively spliced forms of Ich-1, Ich-1L and Ich-1S, were amplified by PCR from an embryonic chicken cDNA library, only the Ich-1L form was found to be expressed in adult ovarian granulosa and theca tissues. The deduced amino acid (aa) sequence of ICH-1L is 70.8% identical to human ICH-1L and contains the conserved QACRG peptide active catalytic sequence characteristic of many ICE-related family of cysteine proteases.

Characterization of the FET4 protein of yeast. Evidence for a direct role in the transport of iron.

The low affinity Fe2+ uptake system of Saccharomyces cerevisiae requires the FET4 gene. In this report, we present evidence that FET4 encodes the Fe2+ transporter protein of this system. Antibodies prepared against FET4 detected two distinct proteins with molecular masses of 63 and 68 kDa. In vitro synthesis of FET4 suggested that the 68-kDa form is the primary translation product, and the 63-kDa form may be generated by proteolytic cleavage of the full-length protein. Consistent with its role as an Fe2+ transporter, FET4 is an integral membrane protein present in the plasma membrane. The level of FET4 closely correlated with uptake activity over a broad range of expression levels and is itself regulated by iron. Furthermore, mutations in FET4 can alter the kinetic properties of the low affinity uptake system, suggesting a direct interaction between FET4 and its Fe2+ substrate. Mutations affecting potential Fe2+ ligands located in the predicted transmembrane domains of FET4 significantly altered the apparent Km and/or Vmax of the low affinity system. These mutations may identify residues involved in Fe2+ binding during transport.

Expression of avian urokinase and tissue-type plasminogen activator messenger ribonucleic acid during follicle development and atresia.

While several studies have documented the presence of plasminogen activator (PA) activity in hen ovarian follicle granulosa and theca tissues, to date it has not been possible to conclusively distinguish between the urokinase (u) and the tissue-type (t) form of the enzyme; this inability is due, in part, to lack of the cloned or characterized chicken tPA gene or gene product. Thus, the present studies were conducted to identify a partial cDNA for chicken tPA and subsequently to evaluate expression of uPA and tPA mRNA in granulosa and theca tissues in vivo and in vitro. Urokinase PA and mRNA levels were highest in prehierarchical-follicle granulosa (3- to 5- and 6- to 8-mm follicles) and theca (6- to 8-mm follicles) tissue compared to hierarchical (9-12 mm through largest preovulatory) follicles. In vitro treatment with a phorbol ester (phorbol 12-myristate, 13-acetate), but not a cAMP analogue (8-bromo-cAMP), significantly increased uPA mRNA levels in both granulosa and theca tissue from the largest and second-largest preovulatory follicles. Of special interest was the finding that levels of uPA mRNA were 10.9-fold higher in atretic compared to morphologically normal 3- to 5-mm follicles. Moreover, 4- to 8-mm-follicle granulosa cells, which spontaneously undergo apoptosis in vitro, demonstrated a rapid increase in uPA mRNA levels after 1 h of incubation (prior to the detection of oligonucleosome formation) while levels in preovulatory-follicle granulosa cells, which do not undergo spontaneous apoptosis, were not altered after 18 h of incubation. By contrast, while tPA mRNA can be identified in granulosa and theca tissues from prehierarchical and preovulatory follicles following polymerase chain reaction amplification, constitutively expressed levels of the transcript were too low to reliably measure by Northern blot analysis. These data indicate that while the chicken expresses a tPA gene that is homologous to the mammalian tPA, uPA is the predominant PA expressed in the hen ovary. In addition, the higher levels of uPA mRNA found in granulosa cells actively undergoing apoptosis and in follicles most susceptible to atresia (4-8 mm) suggest a role for this protease in mediating processes both during the early stages of programmed cell death and in the later stages of follicle atresia and resorption.