Molecular cloning of porcine (Sus scrofa) tumor necrosis factor receptor 2.

Tumor necrosis factor (TNF) alpha can induce both cell death and proliferation by binding to either TNF receptor (TNFR) 1 or 2. In the granulosa cells of porcine ovaries, TNFalpha is considered to act as an anti-apoptotic/survival factor during follicular atresia. As a first step toward elucidating the function of TNFR2 in regulating follicular development/growth and atresia in porcine ovaries, we isolated the porcine (Sus scrofa) cDNA encoding TNFR2, which was identified from a cDNA library prepared from the follicular granulosa cells of pig ovaries. Porcine TNFR2 (1,125 bp, 375 amino acid residues), which contains specific amino acid region of transmembrane, indicated high identities with human and murine TNFR2 (78 and 69% at mRNA level, respectively; 73 and 61% at protein level, respectively), suggesting that the function of porcine TNFR2 is similar to that of human and murine homologues. Understanding the expression patterns of porcine TNFR2 mRNA in various organs, which we confirmed by reverse transcription polymerase chain reaction analysis, would help to elucidate the physiological role of TNFR2 in the regulation of apoptosis in porcine organs.

Anti-apoptotic activity of porcine cFLIP in ovarian granulosa cell lines.

In mammalian ovaries, more than 99% of follicles undergo atresia during growth and development. Recently, we found that the expression of cellular FLICE-like inhibitory protein long form (cFLIP(L)) decreased during follicular atresia in granulosa cells of porcine ovaries. In humans and other species, both the short (cFLIP(S)) and long (cFLIP(L)) forms of cFLIP are considered to function as cell survival factors that inhibit death ligand receptor-mediated apoptosis. Since the anti-apoptotic activity of porcine cFLIP (pcFLIP) in granulosa cells had not been determined, we examined the effect of pcFLIP on survival using granulosa-derived cell lines. A human cervix adenocarcinoma cell line, HeLa, human ovarian granulosa tumor cell line, KGN, and porcine granulosa-derived cell line, JC-410, were used. By Western blotting, internal cFLIP(L) was detected in all cell lines, but only trace levels of cFLIP(S) were found in HeLa and KGN cells. To examine the anti-apoptotic activity, pcFLIP(S) or pcFLIP(L) was overexpressed in HeLa and KGN cells. Transfected cells in which pcFLIP(S) or pcFLIP(L) was overexpressed, survived the induction of Fas-mediated apoptosis, while almost all of the cells transfected with empty vector died. Then, we suppressed the expression of porcine cFLIP(S) and/or cFLIP(L) in JC-410 cells using small interfering RNA (siRNA). When both cFLIP(S) and cFLIP(L), or only cFLIP(L) was suppressed, cell viability declined significantly. From the results, we conclude that porcine cFLIP(S) and cFLIP(L) exhibit anti-apoptotic activity in granulosa-derived cells. It was strongly suggested that pcFLIP acts as a survival-promoting factor in granulosa cells and determines whether porcine ovarian follicles survive or undergo atresia.

Changes in expression of interleukin-6 receptors in granulosa cells during follicular atresia in pig ovaries.

More than 99% of follicles undergo a degenerative process known as "atresia" in mammalian ovaries, and only a few follicles ovulate during follicular growth and development. Follicular selection predominantly depends on granulosa cell apoptosis. To reveal the molecular mechanisms of selective follicular atresia, we examined the changes in the levels of interleukin-6 (IL-6) receptors expressed in the granulosa cells of pig ovaries. The levels of IL-6 receptor (IL-6R)-alpha mRNA and protein in granulosa cells were quantified by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, respectively. IL-6R alpha mRNA and protein were highly expressed in the granulosa cells of progressed atretic follicles. Enzyme-linked immunosorbent assay showed that the expression of IL-6 soluble receptor (IL-6sR) protein in follicular fluid decreased during atresia. Moreover, we isolated porcine cDNA encoding an IL-6 signal transducer, gp130. Porcine gp130 (2,754 bp and 917 amino acids) was identified from a cDNA library prepared using follicular granulosa cells of pig ovaries. Porcine gp130 was highly homologous with human and murine gp130. RT-PCR analysis revealed that the level of gp130 mRNA also decreased during atresia. We presume that IL-6sR and gp130, but not IL-6R alpha, play important roles in regulation of granulosa cell survival.

The role of interleukin-6 in the regulation of granulosa cell apoptosis during follicular atresia in pig ovaries.

More than 99% of follicles in mammalian ovaries undergo a degenerative process known as atresia, and only a few follicles actually ovulate during follicular growth and development. Follicular selection mostly depends on granulosa cell apoptosis, but the molecular mechanism behind the regulation of this selective atresia is still largely unknown. In the present study, to examine whether or not interleukin-6 (IL-6), a multifunctional cytokine, is involved in apoptosis during atresia in pig ovaries, the expression of IL-6 mRNA in granulosa cells was quantified by real-time reverse transcription-polymerase chain reaction (RT-PCR). The level of mRNA decreased during atresia. Enzyme-linked immunosorbent assay (ELISA) showed that the level of IL-6 protein in follicular fluid also decreased during atresia. Moreover, recombinant human IL-6 upregulated the expression of an intracellular apoptosis inhibitor, cellular FLICE-like inhibitory protein long form (cFLIP(L)), in cultured cells derived from human granulosa cells. It is possible that IL-6 is produced in the granulosa cells of healthy follicles, that it increases the cFLIP(L) level, and cFLIP(L) then prevents apoptotic cell death.

Molecular characteristics of porcine Fas-associated death domain (FADD) and procaspase-8.

To reveal the intracellular signal transduction molecules involved in granulosa cell apoptosis in porcine ovarian follicles, we cloned the porcine Fas-associated death domain (FADD), an adaptor protein for the cell death receptor, and procaspase-8, an initiator caspase. Porcine FADD (pFADD) was 636 bp (211 amino acids: aa) long and showed 74.0 and 65.4% homology with human and murine FADD, respectively. Porcine procaspase-8 (pprocaspase-8) was 1,431 bp (476 aa) long and 70.6 and 63.4% homologous with human and murine procaspase-8, respectively. To confirm the apoptosis-inducing abilities, we constructed pFADD and pprocaspase-8 cDNA expression vectors with enhanced green fluorescence protein (EGFP) and then transfected them into human uterine cervix tumor (HeLa-K), human granulosa cell-derived (KGN), murine granulosa-derived tumor (KK1), and porcine granulosa cell-derived (JC410) cells. When pFADD and pprocaspase-8 were overexpressed, cell death was induced in these transfected cells. However when caspase-inhibitor p35 was cotransfected, cell death was inhibited. The pFADD and pprocaspase-8 genes are well conserved, as are the physiological functions of their products.

Expression and localization of Fas ligand and Fas during atresia in porcine ovarian follicles.

To reveal the mechanisms regulating the selective atresia of follicles in porcine ovaries, we examined the changes in the mRNA and protein levels of cell-death ligand, Fas/APO-1/CD95 ligand (FasL), and its receptor, Fas/APO-1/CD95 (Fas), and the localization of the proteins in granulosa cells during follicular atresia using the reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemical techniques, respectively. Trace levels of FasL mRNA and protein were detected in the granulosa cells of healthy follicles; however, weak levels were detected in those of early atretic follicles, and the levels increased during atresia. Trace/weak levels of Fas mRNA and protein were detected in the granulosa cells of healthy follicles. Fas protein was located in the cytoplasmic area, not in cell membrane area, indicating that it has no activity in regard to inducing apoptosis. When apoptosis commences in granulosa cells, Fas moves from the cytoplasmic to cell membrane area. FasL and Fas mRNAs and proteins in granulosa cells were upregulated during follicular atresia. The FasL and Fas system may play a crucial role in the regulation of apoptosis in granulosa cells during selective follicular atresia in porcine ovaries.

The regulation of ovarian granulosa cell death by pro- and anti-apoptotic molecules.

In the mammalian ovary, follicular development and atresia are closely regulated by cell death and survival-promoting factors, including hormones (gonadotropins) and intraovarian regulators (gonadal steroids, cytokines, and intracellular proteins). Several hundred thousand primordial follicles are present in the mammalian ovary; however, only a limited number of primordial follicles develop to the preovulatory stage and ovulate. The others, more than 99% of follicles, will be eliminated via a degenerative process known as "atresia". The endocrinological regulatory mechanisms involved in follicular development and atresia have been characterized to a large extent, but the precise temporal and molecular mechanisms involved in the regulation of these events have remained largely unknown. Recent studies suggest that the apoptosis of ovarian granulosa cells plays a major role in follicular atresia. In this review, we provide an overview of development and atresia of follicles, and apoptosis of granulosa cells in mammals.

Regulation of the ovine interferon-tau gene by a blastocyst-specific transcription factor, Cdx2.

Expression of ovine interferon-tau (oIFNtau), a factor essential for the process of maternal recognition of pregnancy in ruminant ungulates, is restricted to the trophoblast. However, the molecular mechanisms by which oIFNtau expression is restricted to the trophectoderm have not been fully elucidated. The objective of this study was to determine whether oIFNtau gene transcription could be regulated through Cdx2 expression, a transcription factor implicated in the control of cell differentiation in the trophectoderm. Human choriocarcinoma JEG3 cells were co-transfected with an oIFNtau (-654 base pair, bp)-luciferase reporter (-654-oIFNtau-Luc) construct and several transcription factor expression plasmids. Compared to -654-oIFNtau-Luc alone, transcription of the -654-oIFNtau-Luc increased more than 30 times when this construct was co-transfected with Cdx2, Ets-2, and c-jun. The degree of transcription decreased to 1/4 levels when the upstream region was reduced to -551 bp, and became minimal with further deletions; this was confirmed with the use of the reporter constructs with mutated c-jun, Ets-2, and/or Cdx2 sites. In trophoblast unrelated NIH3T3 cells, which do not support IFNtau gene transcription, the oIFNtau-Luc transcription was enhanced approximately eightfold when the cells were co-transfected with the Cdx2/Ets-2 or Cdx2/Ets-2/c-jun expression plasmids. These findings were confirmed by gel-shift assays examining Cdx binding site on the oIFNtau gene's upstream region, by immunohistochemical study identifying the presence of Cdx2 in day 15 and 17 ovine conceptuses, and by Western blot detecting Cdx2 in day 17 conceptuses. Our results indicate that oIFNtau gene transcription is regulated by Cdx2, and suggest that Cdx2 could be a key molecule in determining oIFNtau gene transcription by the trophectoderm.

Changes in expression and localization of connexin 43 mRNA and protein in porcine ovary granulosa cells during follicular atresia.

Gap junctions contain channels that connect neighboring cells by allowing the movement of molecules smaller than 1,200 Da. They are formed by connexins and may play a crucial role in the regulation of apoptotic cell death. To determine the role of connexin 43 (Cx43), which is dominantly expressed in granulosa cells, in the regulation of granulosa cell apoptosis during follicular atresia, we examined the changes in the expression and localization of Cx43 mRNA and protein in granulosa cells during atresia using the quantitative real-time revese transcription-polymerase chain reaction, in situ hybridization, Western blot, and immunohistochemistry. Stages of follicular atresia were assessed based on histochemical terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick end-labeling (TUNEL) and/or the ratio of progesterone and 17beta-estradiol levels in follicular fluid measured by radioimmunoassay. Cx43 mRNA was detected in granulosa cells of secondary follicles and of healthy, early and progressed atretic tertiary follicles, but not in those of primordial or primary follicles. Both phosphorylated/activated and non-phosphorylated/native Cx43 proteins were detected in granulosa cells of secondary and tertiary follicles, but not in those of primordial or primary follicles. Moreover, in tertiary follicles, these Cx43 proteins were expressed most strongly in granulosa cells of healthy follicles, but only trace levels were noted in cells of early atretic and progressed atretic follicles, an indication that the expression levels of Cx43 protein decrease during follicular atresia. These findings indicate that Cx43 is involved in the apoptosis of granulosa cells during atresia in porcine ovaries.

Changes in expression of anti-apoptotic protein, cFLIP, in granulosa cells during follicular atresia in porcine ovaries.

Follicular selection is performed in mammalian ovaries, as most follicles undergo atresia during follicular development and growth. Follicular regression is indicated to begin with granulosa cell apoptosis. To reveal the molecular mechanisms of the selection, we examined the changes in the levels of cellular-Flice like inhibitory protein (cFLIP) expression in porcine granulosa cells. cFLIP is the homologue of intracellular apoptosis inducer (procaspase-8/Flice), and has two alternative splicing isoforms: cFLIP short form (cFLIP(S)) and long form (cFLIP(L)). By competing with caspase-8, cFLIP inhibits apoptosis initiated by death receptors. The changes in the levels of cFLIP(S) and cFLIP(L) mRNA and protein expression in granulosa cells were determined by RT-PCR and Western blotting, respectively. cFLIP(L) mRNA and protein were highly expressed in granulosa cells of healthy follicles and decreased during atresia. cFLIP(S) mRNA levels in granulosa cells were low and showed no change among the stages of follicular development, and its protein level was extremely low. We examined the changes in the localization of cFLIP mRNAs in pig ovaries by in situ hybridization and found that cFLIP(L) is abundant in granulosa cells of healthy follicles in comparison with those of atretic follicles. Immunohistochemical analyses demonstrated that the cFLIP protein is highly expressed in the granulosa cell of healthy follicles but weakly expressed in that of atretic follicles. We presumed that cFLIP, especially cFLIP(L), plays an anti-apoptotic role in the granulosa cells of healthy follicles of pig ovaries, and that cFLIP could be a major survival factor that determines whether growth or atresia occurs in porcine follicles.

Different levels of ovine interferon-tau gene expressions are regulated through the short promoter region including Ets-2 binding site.

Regulation of interferon-tau (IFNtau) production, a conceptus secretory protein implicated in the process of maternal recognition of pregnancy, has not been fully elucidated. Among more than 10 ovine IFNtau (oIFNtau) gene sequences characterized, approximately 75% of oIFNtau transcripts expressed in utero is derived from oIFNtau-o10 gene and amounts of transcripts from other oIFNtau genes such as oIFNtau-o8 or oIFNtau-o2 are minimal. It was hypothesized that the variation in expression levels exhibited by oIFNtau-o10 and oIFNtau-o8/-o2 genes was due to differences in the proximal promoter regions of these oIFNtau genes. To test this hypothesis, transient transfection experiments with human choriocarcinoma JEG3 cells were executed with deleted and/or mutated 5'-upstream regions of these oIFNtau genes attached to the chloramphenicol acetyltransferase (CAT) reporter gene. Because only the Ets-2 binding site located in the oIFNtau-o10 gene appeared to differentiate the expression levels of these constructs, the 6 base pair (bp) Ets-2 sequence from the oIFNtau-o10 gene inserted into the oIFNtau-o8/-o2 gene-reporter construct was examined. The insertion of this Ets-2 binding site into the oIFNtau-o8/o2-reporter construct failed to increase the degree of transactivation. Rather than this 6 bp sequence, a 22 bp sequence of the proximal promoter region, including the Ets-2 binding site, of the oIFNtau-o10 gene was required for oIFNtau-o8/-o2-reporter transactivation. By electrophoretic mobility shift assay (EMSA), nuclear protein(s) bound to this 22 bp from the oIFNtau-o10 and oIFNtau-o8/o2 genes differed. These results suggest that the short promoter region including the Ets-2 binding site, not the Ets-2 binding region itself, may determine different levels of oIFNtau gene expressions seen in utero.

Regulation mechanism of selective atresia in porcine follicles: regulation of granulosa cell apoptosis during atresia.

More than 99% of follicles undergo a degenerative process known as "atresia", in mammalian ovaries, and only a few follicles ovulate during ovarian follicular development. We have investigated the molecular mechanism of selective follicular atresia in mammalian ovaries, and have reported that follicular selection dominantly depends on granulosa cell apoptosis. However, we have little knowledge of the molecular mechanisms that control apoptotic cell death in granulosa cells during follicle selection. To date, at least five cell death ligand-receptor systems [tumor necrosis factor (TNF)alpha and receptors, Fas (also called APO-1/CD95) ligand and receptors, TNF-related apoptosis-inducing ligand (TRAIL; also called APO-2) and receptors, APO-3 ligand and receptors, and PFG-5 ligand and receptors] have been reported in granulosa cells of porcine ovaries. Some cell death ligand-receptor systems have "decoy" receptors, which act as inhibitors of cell death ligand-induced apoptosis in granulosa cells. Moreover, we showed that the porcine granulosa cell is a type II apoptotic cell, which has the mitochondrion-dependent apoptosis-signaling pathway. Briefly, the cell death receptor-mediated apoptosis signaling pathway in granulosa cells has been suggested to be as follows. (1) A cell death ligand binds to the extracellular domain of a cell death receptor, which contains an intracellular death domain (DD). (2) The intracellular DD of the cell death receptor interacts with the DD of the adaptor protein (Fas-associated death domain: FADD) through a homophilic DD interaction. (3) FADD activates an initiator caspase (procaspase-8; also called FLICE), which is a bipartite molecule, containing an N-terminal death effector domain (DED) and a C-terminal DD. (4) Procaspase-8 begins auto-proteolytic cleavage and activation. (5) The auto-activated caspase-8 cleaves Bid protein. (6) The truncated Bid releases cytochrome c from mitochondrion. (7) Cytochrome c and ATP-dependent oligimerization of apoptotic protease-activating factor-1 (Apaf-1) allows recruitment of procaspase-9 into the apoptosome complex. Activation of procaspase-9 is mediated by means of a conformational change. (8) The activated caspase-9 cleaves downstream effector caspases (caspase-3). (9) Finally, apoptosis is induced. Recently, we found two intracellular inhibitor proteins [cellular FLICE-like inhibitory protein short form (cFLIPS) and long form (cFLIPL)], which were strongly expressed in granulosa cells, and they may act as anti-apoptotic/survival factors. Further in vivo and in vitro studies will elucidate the largely unknown molecular mechanisms, e. g. which cell death ligand-receptor system is the dominant factor controlling the granulosa cell apoptosis of selective follicular atresia in mammalian ovaries. If we could elucidate the molecular mechanism of granulosa cell apoptosis (follicular selection), we could accurately diagnose the healthy ovulating follicles and precisely evaluate the oocyte quality. We hope that the mechanism will be clarified and lead to an integrated understanding of the regulation mechanism.

Porcine (Sus scrofa) cellular FLICE-like inhibitory protein (cFLIP): molecular cloning and comparison with the human and murine cFLIP.

To reveal the molecular regulation mechanism of selective follicular atresia in porcine ovaries, we isolated the porcine cDNA encoding cellular FLICE-like inhibitory protein (cFLIP), which inhibits death receptor-mediated apoptosis signal transduction. Two alternative splicing isoforms of cFLIP, porcine cellular FLIP-short form (pcFLIPS, 642 bp and 214-aa) and -long form (pcFLIPL, 1446 bp and 482-aa), were identified from a cDNA library prepared from follicular granulosa cells of pig ovaries. pcFLIPS and pcFLIPL indicated high identities with human and murine cFLIP, and both of them contain two tandem specific amino acid regions (death effector domain: DED) in their N-terminal, suggesting that pcFLIPS and pcFLIPL inhibit the death receptor-mediated apoptosis signal by binding to other pro-apoptotic factors mediated by DED. pcFLIPS contains a short C-terminal region, while pcFLIPL has a caspase-like domain in the C-terminal region. The reverse transcription-polymerase chain reaction analysis revealed that both pcFLIPS and pcFLIPL mRNAs were highly expressed in granulosa cells of healthy follicles, suggesting that these cFLIPs play important roles in the regulation mechanism of apoptosis in ovarian follicular granulosa cells. The present data will contribute to understanding of the physiological roles of cFLIPs in the apoptosis regulation in porcine tissues.