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.

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.

Associations between DNA sequence variation and variation in expression of the Adh gene in natural populations of Drosophila melanogaster.

A large part of the genetic variation in alcohol dehydrogenase (ADH) activity level in natural populations of Drosophila melanogaster is associated with segregation of an amino acid replacement polymorphism at nucleotide 1490, which generates a difference in electrophoretic mobility. Part of the allozymic difference in activity level is due to a catalytic efficiency difference, which is also caused by the amino acid replacement, and part is due to a difference in the concentration of ADH protein. A previous site-directed in vitro mutagenesis experiment clearly demonstrated that the amino acid replacement has no effect on the concentration of ADH protein, nor does a strongly associated silent polymorphism at nucleotide 1443. Here we analyze associations between polymorphisms within the Adh gene and variation in ADH protein level for a number of chromosomes derived from natural populations. A sequence length polymorphism within the first intron of the distal (adult) transcript, 1, is in strong linkage disequilibrium with the amino acid replacement. Among a sample of 46 isochromosomal lines analyzed, all but one of the 14 Fast lines have 1 and all but one of the 32 Slow lines lack 1. The exceptional Fast line has an unusually low level of ADH protein (typical of Slow lines) and the exceptional Slow line has an unusually high level (typical of Fast lines). These results suggest that the 1 polymorphism may be responsible for the average difference in ADH protein between the allozymic classes. A previous experiment localized the effect on ADH protein to a 2.3-kb restriction fragment. DNA sequences of this fragment from several alleles of each allozymic type indicate that no other polymorphisms within this region are as closely associated with the ADH protein level difference as the 1 polymorphism.

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.

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.

Induction of apoptotic cell death in hen granulosa cells by ceramide.

Recent studies have demonstrated that ovarian follicle atresia occurs extensively before follicle selection into the avian preovulatory hierarchy, and that this process is mediated via granulosa cell apoptosis. Subsequent to follicle selection, granulosa cells are inherently resistant to apoptosis, and such resistance is correlated with increased expression of death suppressor genes such as bcl-xlong. In the present studies we used this avian ovary model system to 1) identify cellular characteristics and mechanisms related to apoptotic cell death of granulosa cells in vitro, and 2) further characterize functional differences between apoptosis-susceptible (4- to 8-mm follicle) and apoptosis-resistant (preovulatory follicle) granulosa cells. Treatment of granulosa cells from the largest preovulatory follicle with N-octanoylsphingosine (C8-ceramide) results in pronounced oligonucleosome formation, a hallmark of apoptosis. That this is indicative of programmed cell death is supported by an increased incidence of pyknotic nuclei and apoptotic bodies in C8-ceramide-treated samples compared to that in control cultured cells. Tumor necrosis factor-alpha, a stimulator of ceramide production, actively promotes oligonucleosome formation in apoptosis-susceptible, but not in apoptosis-resistant, granulosa cells. Induction of apoptosis is also observed after exposure of apoptosis-resistant granulosa cells to sphingomyelinase treatment and UV irradiation, which are known to stimulate endogenous ceramide production, and to the anticancer drug, daunorubicin, which initiates de novo ceramide biosynthesis via activation of ceramide synthase. Although treatment of granulosa cells with fumonisin B1, a specific ceramide synthase inhibitor, blocks daunorubicin-stimulated oligonucleosome formation, UV-induced cell death is unaffected. Taken together, these results demonstrate that pharmacological factors known to mimic the actions of ceramide or stimulate ceramide production can induce oligonucleosome formation and programmed cell death in granulosa cells. More importantly, however, the ability of a physiologically relevant initiator of ceramide biosynthesis, tumor necrosis factor-alpha, to promote cell death is evident only in apoptosis-susceptible granulosa cells collected from atresia-prone prehierarchal follicles. These data provide support for ceramide as an important intracellular signaling mechanism, mediating granulosa cell apoptosis and follicle atresia.

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.

Susceptibility of avian ovarian granulosa cells to apoptosis is dependent upon stage of follicle development and is related to endogenous levels of bcl-xlong gene expression.

Studies were conducted to evaluate the susceptibility of avian ovarian granulosa cells to apoptosis when incubated in vitro and to relate this relative susceptibility to both the stage of follicle development from which granulosa cells were collected (atresia-prone vs. -resistant) and to the expression of a gene previously linked to the regulation of cell viability, bcl-xlong. Granulosa cells from slow growing, prehierarchal (4- to 8-mm diameter; atresia-prone) follicles were found to undergo rapid and progressively extensive apoptosis after incubation in defined medium for 6-24 h (P < 0.05 vs. unincubated controls). By contrast, cells from the largest preovulatory (F1) follicle, as well as from follicles most recently recruited into the follicle hierarchy (9- to 12-mm diameter), showed significantly less low molecular wt labeling at 6 h of incubation (P < 0.05 vs. 4- to 8-mm follicles). Furthermore, the amount of low molecular wt labeling did not significantly increase in cells from either stage of follicle development at 12 or 24 h of incubation (P < 0.05 vs. 6 h incubation). This biochemical indication of ongoing apoptosis in prehierarchal follicle granulosa cells was confirmed by an increased incidence of pyknotic nuclei detected by morphological analysis. Thus, increased susceptibility to apoptosis in incubated prehierarchal follicle granulosa cells is correlated with the high rate of follicle atresia that is known to occur at this stage of development in vivo. Recombinant human FSH (100 mIU) and transforming growth factor-alpha (3.3 nM) partially suppressed apoptosis in prehierarchal follicle granulosa cells after 6 h of incubation (by 46-57%; P < 0.05 vs. control), as did the cAMP analog, 8-Br-cAMP (1 mM; by 59%; P < 0.05). A single form of the bcl-2-related gene, bcl-x, was detected in hen ovarian tissues; this transcript corresponded to bcl-xlong, the death-suppressing form of bcl-x. The highest levels of bcl-xlong messenger RNA were found in granulosa tissue from preovulatory follicles, with significantly lower levels detected in prehierarchal follicle granulosa tissue (P < 0.05). Elevated expression of bcl-xlong in preovulatory follicles was correlated to increased resistance to the process of apoptosis, in vitro, and the virtual absence of follicle atresia at this stage of development, in vivo. We conclude that there is a direct relationship between the inherent susceptibility of avian granulosa cells to apoptosis and the high rate of follicle atresia in follicles not yet selected into the preovulatory hierarchy. Moreover, our results are consistent with the proposal that the expression of death-suppressing genes, including bcl-xlong, is capable of rendering cells resistant to the process of apoptosis. The findings reported herein provide the foundation for a novel model with which to further elucidate molecular mechanisms related not only to the initiation of follicle atresia, but also events associated with the process of follicle selection.

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 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.

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.

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.

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.

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.

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.

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.

Characterization of a chicken luteinizing hormone receptor (cLH-R) complementary deoxyribonucleic acid, and expression of cLH-R messenger ribonucleic acid in the ovary.

Studies were conducted to characterize the chicken ovarian LH receptor (cLH-R) cDNA and to evaluate expression of cLH-R mRNA in follicles at different stages of development. A total of 1.89 kb of nucleic acid sequence corresponding to the cLH-R (1.79 kb of the predicted coding region) was isolated by a combination of reverse transcription-polymerase chain reaction and cDNA library screening techniques. Also of interest was the finding that two of three positive clones isolated from the hen ovarian cDNA library contained an 86-bp insert located in the extracellular domain within 69 bp of the putative transmembrane domain. This insert contains an inframe TGA stop codon, suggesting that an alternatively spliced transcript results in translation of a truncated protein corresponding to the extracellular domain of the cLH-R. Considering all protein domains thus far characterized, the deduced amino acid sequence of the cLH-R shares 73.2% and 74.2% identity with the rat and porcine LH-R sequences, respectively, with highest homology occurring within the seven transmembrane spanning regions (86-88% identity vs. mammalian sequences). Northern blot analysis determined that cLH-R mRNA levels in the theca layer tend to increase through follicle development to the second largest (F2) preovulatory follicle (p = 0.084), and to decrease in the largest preovulatory (F1) follicle (p < 0.02 vs. F2). By comparison, cLH-R mRNA levels are nondetectable (by Northern blot analysis) in granulosa cells from prehierarchal (3-8-mm diameter) follicles. Constitutive expression of cLH-R mRNA in granulosa cells is first detectable at the 9-12-mm diameter stage of follicle development, and levels are further increased in cells from large preovulatory (F1, F2, and F3) follicles (p < 0.01 vs. 9-12-mm stage). Collectively, these results are consistent with previous observations that granulosa cells from prehierarchal follicles fail to produce cAMP or steroids in response to short-term incubation with ovine LH, in vitro, and that granulosa cells acquire LH responsiveness only subsequent to follicle selection into the rapid growth phase.

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.