Context-dependent modulation of Pol II CTD phosphatase SSUP-72 regulates alternative polyadenylation in neuronal development.

Alternative polyadenylation (APA) is widespread in neuronal development and activity-mediated neural plasticity. However, the underlying molecular mechanisms are largely unknown. We used systematic genetic studies and genome-wide surveys of the transcriptional landscape to identify a context-dependent regulatory pathway controlling APA in the Caenorhabditis elegans nervous system. Loss of function in ssup-72, a Ser5 phosphatase for the RNA polymerase II (Pol II) C-terminal domain (CTD), dampens transcription termination at a strong intronic polyadenylation site (PAS) in unc-44/ankyrin yet promotes termination at the weak intronic PAS of the MAP kinase dlk-1. A nuclear protein, SYDN-1, which regulates neuronal development, antagonizes the function of SSUP-72 and several nuclear polyadenylation factors. This regulatory pathway allows the production of a neuron-specific isoform of unc-44 and an inhibitory isoform of dlk-1. Dysregulation of the unc-44 and dlk-1 mRNA isoforms in sydn-1 mutants impairs neuronal development. Deleting the intronic PAS of unc-44 results in increased pre-mRNA processing of neuronal ankyrin and suppresses sydn-1 mutants. These results reveal a mechanism by which regulation of CTD phosphorylation controls coding region APA in the nervous system.

Expression of tissue factor and tissue factor pathway inhibitors during ovulation in rats: a relevance to the ovarian hyperstimulation syndrome.

BACKGROUND: Blood coagulation has been associated with ovulation and female infertility. In this study, the expression of the tissue factor system was examined during ovulation in immature rats; the correlation between tissue factor and ovarian hyperstimulation syndrome (OHSS) was evaluated both in rats and human follicular fluids. METHODS: Ovaries were obtained at various times after human chorionic gonadotropin (hCG) injection to investigate the expression of tissue factor system. Expression levels of ovarian tissue factor, tissue factor pathway inhibitor (Tfpi)-1 and Tfpi-2 genes and proteins were determined by real-time quantitative polymerase chain reaction (qPCR), and Western blot and immunofluorescence analyses, respectively. Expression levels of tissue factor system were also investigated in ovaries of OHSS-induced rats and in follicular fluid of infertile women. RESULTS: The expression of tissue factor in the preovulatory follicles was stimulated by hCG, reaching a maximum at 6 h. Tissue factor was expressed in the oocytes and the preovulatory follicles. Tfpi-2 mRNA levels were mainly increased by hCG in the granulosa cells whereas the mRNA levels of Tfpi-1 were decreased by hCG. Human CG-stimulated tissue factor expression was inhibited by the progesterone receptor antagonist. The increase in Tfpi-2 expression by hCG was decreased by the proliferator-activated receptor γ (PPARγ) antagonist. Decreased expression of the tissue factor was detected in OHSS-induced rats. Interestingly, the tissue factor concentrations in the follicular fluids of women undergoing in vitro fertilization were correlated with pregnancy but not with OHSS. CONCLUSIONS: Collectively, the results indicate that tissue factor and Tfpi-2 expression is stimulated during the ovulatory process in rats; moreover, a correlation exists between the levels of tissue factor and OHSS in rats but not in humans.

Involvement of peroxiredoxin 2 in cumulus expansion and oocyte maturation in mice.

Peroxiredoxin 2 (Prdx2), an antioxidant enzyme, is expressed in the ovary during the ovulatory process. The aim of the present study was to examine the physiological role of Prdx2 during ovulation using Prdx2-knockout mice and mouse cumulus-oocyte complex (COC) from WT mice. Two days of treatment of immature mice (21-23 days old) with equine chorionic gonadotrophin and followed by treatment with human chorionic gonadotrophin greatly impaired cumulus expansion and oocyte maturation in Prdx2-knockout but not wild-type mice. Treatment of COCs in culture with conoidin A (50µM), a 2-cys Prdx inhibitor, abolished epiregulin (EPI)-induced cumulus expansion. Conoidin A treatment also inhibited EPI-stimulated signal molecules, including signal transducer and activator of transcription-3, AKT and mitogen-activated protein kinase 1/2. Conoidin A treatment also reduced the gene expression of EPI-stimulated expansion-inducing factors (hyaluronan synthase 2 (Has2), pentraxin 3 (Ptx3), TNF-α induced protein 6 (Tnfaip6) and prostaglandin-endoperoxide synthase 2 (Ptgs2)) and oocyte-derived factors (growth differentiation factor 9 (Gdf9) and bone morphogenetic protein 15 (Bmp15)). Furthermore, conoidin A inhibited EPI-induced oocyte maturation and the activity of connexins 43 and 37. Together, these results demonstrate that Prdx2 plays a role in regulating cumulus expansion and oocyte maturation during the ovulatory process in mice, probably by modulating epidermal growth factor receptor signalling.

Peroxiredoxins are required for spindle assembly, chromosome organization, and polarization in mouse oocytes.

Peroxiredoxins (Prxs) are highly conserved antioxidant enzymes and are implicated in multiple biological processes; however, their function in oocyte meiosis has not been studied. Here we show that inhibition of Prx I and II results in spindle defects, chromosome disorganization, and impaired polarization in mouse oocytes. Prx I was specifically localized at the spindle, whereas Prx II was enriched at the oocyte cortex and chromosomes. Inhibition of Prx activity with conoidin A disturbed assembly of the microtubule organizing center (MTOC) through Aurora A regulation, leading to defects in spindle formation. Moreover, conoidin A impaired actin filament and cortical granule (CG) distribution, disrupting actin cap and CG formation, respectively. Conoidin A also increased DNA damage without significantly increasing reactive oxygen species (ROS) levels, suggesting that the effects of conoidin A on meiotic maturation are not likely associated with ROS scavenging pathways. Therefore, our data suggest that Prxs are required for spindle assembly, chromosome organization, and polarization during meiotic maturation.

Regulation of interleukin-11 expression in ovulatory follicles of the rat ovary.

The aim of the present study was to examine the regulation of interleukin (IL)-11 expression, as well as the role of IL-11, during ovulation in gonadotropin-primed immature rats. Injection of equine chorionic gonadotropin (eCG), followed by human CG (hCG) to induce superovulation stimulated expression of the Il11 gene in theca cells within 6h, as revealed by northern blot and in situ hybridisation analyses. Real-time reverse transcription-polymerase chain reaction analysis showed that the IL-11 receptor, α subunit gene was expressed in granulosa and theca cells and that injection of hCG had no effect on its expression. IL-11 protein expression was stimulated in theca cells by hCG. LH-stimulated increases in Il11 mRNA levels in cultured preovulatory follicles were inhibited by protein kinase A and mitogen-activated protein kinase kinase inhibitors. Toll-like receptor (TLR) 2 and TLR4 were detected in preovulatory follicles, and the TLR4 ligand lipopolysaccharide, but not the TLR2 ligand Pam3Cys, increased Il11 mRNA levels in theca cells, but not in granulosa cells. Treatment of preovulatory follicles with IL-11 stimulated progesterone production and steroidogenic acute regulatory protein (Star) gene expression. Together, these results indicate that IL-11 in theca cells is stimulated by mitogen-activated protein kinase signalling and TLR4 activation, and increases progesterone production during ovulation.

Regulation of uridine diphosphate-glucuronosyltransferase 2B15 expression during ovulation in the rat.

Uridine diphosphate-glucuronosyltransferase 2B15 (UGT2B15) conjugates 5α-androstane-3α, 17ß-diol (3α-diol) to 3α-diol glucuronide (3α-diol G) in steroid target tissues. The present study investigated the regulation of UGT2B15 expression during the ovulatory process in the rat. Real-time PCR analysis revealed that treatment of immature rats with equine chorionic gonadotropin followed by human chorionic gonadotropin transiently stimulated UGT2B15 gene expression in granulosa cells of preovulatory follicles within 6 h. The progesterone receptor antagonist RU486 suppressed the gonadotropin-induced UGT2B15 expression. The expression of UGT2B15 and the levels of 3α-diol G were transiently increased by luteinizing hormone (LH) treatment in cultured preovulatory follicles. The LH-stimulated UGT2B15 mRNA level in cultured preovulatory follicles was inhibited by inhibitors of adenylyl cyclase, phosphoinositide 3-kinase and mitogen-activated protein kinase. Furthermore, a vitamin D receptor agonist (calcitriol) suppressed the LH-stimulated UGT2B15 expression in a dose-dependent manner. Taken together, these results indicate that gonadotropins transiently stimulate UGT2B15 expression and activity in preovulatory follicles, and UGT2B15 mRNA levels are regulated by the progesterone receptor and vitamin D receptor.

Expression of aldo-keto reductase family 1, member C14 during ovulation in the rat.

The potent androgen 5α-dihydrotestosterone is metabolized to the weak androgen 5α-androstane-3α, 17ß-diol (3α-diol) by the enzyme aldo-keto reductase family 1, member C14 (Akr1c14) in rodents. The purpose of the present study was to investigate the regulation of Akr1c14 expression during the ovulatory process in rat ovaries. Northern blot analysis revealed that treatment of immature rats with equine chorionic gonadotropin resulted in lowered Akr1c14 expression, whereas subsequent treatment with human chorionic gonadotropin (hCG) increased ovarian Akr1c14 expression within 3 h. In situ hybridization analysis showed that Akr1c14 mRNA was localized in granulosa cells of growing follicles before hCG treatment, but it was also expressed in granulosa cells of preovulatory follicles after hCG treatment. Akr1c14 protein expression increased after 6 h of hCG treatment and was sustained at high levels until 12 h. The levels of 3α-diol in preovulatory follicles isolated from ovaries in vivo were fluctuated by hCG treatment; decreased at 6 h and increased at 9 h. Human CG-induced Akr1c14 expression was suppressed by treatment with the progesterone receptor antagonist RU486, but not with the cyclooxygenase inhibitor indomethacin. Taken together, these findings demonstrate the induction of Akr1c14 by hCG in granulosa cells of rat preovulatory follicles that was regulated by progesterone receptor antagonist.

A conserved upstream motif orchestrates autonomous, germline-enriched expression of Caenorhabditis elegans piRNAs.

Piwi-interacting RNAs (piRNAs) fulfill a critical, conserved role in defending the genome against foreign genetic elements. In many organisms, piRNAs appear to be derived from processing of a long, polycistronic RNA precursor. Here, we establish that each Caenorhabditis elegans piRNA represents a tiny, autonomous transcriptional unit. Remarkably, the minimal C. elegans piRNA cassette requires only a 21 nucleotide (nt) piRNA sequence and an ∼50 nt upstream motif with limited genomic context for expression. Combining computational analyses with a novel, in vivo transgenic system, we demonstrate that this upstream motif is necessary for independent expression of a germline-enriched, Piwi-dependent piRNA. We further show that a single nucleotide position within this motif directs differential germline enrichment. Accordingly, over 70% of C. elegans piRNAs are selectively expressed in male or female germline, and comparison of the genes they target suggests that these two populations have evolved independently. Together, our results indicate that C. elegans piRNA upstream motifs act as independent promoters to specify which sequences are expressed as piRNAs, how abundantly they are expressed, and in what germline. As the genome encodes well over 15,000 unique piRNA sequences, our study reveals that the number of transcriptional units encoding piRNAs rivals the number of mRNA coding genes in the C. elegans genome.

Cumulus cell-expressed type I interferons induce cumulus expansion in mice.

Ovulation resembles the inflammatory response. The purpose of the present study was to examine the expression and role of type I interferons (IFNs) Ifnalpha and Ifnbeta in mouse ovaries during the process of ovulation. An in vivo injection of equine chorionic gonadotropin (CG)-human CG (hCG) stimulated Ifnalpha and Ifnbeta mRNA in cumulus-oocyte complexes (COCs) within 6 h. Type I IFN receptor (Ifnar1 and Ifnar2) genes were also expressed in preovulatory follicles without a change by hCG. Immunofluorescent study revealed the expression of protein signals of Ifnalpha, Ifnbeta, and Ifnar1 in cumulus cells. Treatment of COCs with Ifnalpha or Ifnbeta in vitro induced cumulus expansion that was comparable to that mediated by epiregulin. In cultured COCs, the levels of Ifnalpha and Ifnbeta mRNA increased by epiregulin and follicle-stimulating hormone, but not by prostaglandin E2. Ifnalpha and Ifnbeta activated multiple signaling events (signal transducer and activator of transcription-1/3, Akt, and mitogen-activated protein kinase 1/2) and stimulated the expression of genes known to impact COC expansion (Has2, Ptx3, Tnfaip6, and Ptgs2). Interestingly, treatment of COCs with Toll-like receptor (TLR) 2 and TLR4 ligands (lipopolysaccharides, Pam3Cys, and hyaluronan fragments) increased Ifnalpha and Ifnbeta mRNA, while coculture with anti-TLR2/4 neutralizing antibody abolished these effects. Taken together, these results demonstrate that the type I IFN system is operating in mouse cumulus cells and plays a role in the induction of cumulus expansion during the ovulatory process in mice.

The novel, actin-like protein Tact3 is expressed in rodent testicular haploid germ cells.

Mouse testis actin-like proteins 1 and 2 (mTact1 and mTact2), which are expressed in murine haploid germ cells, have been described previously. Here, we report the cloning and characterization of a third actin-like protein from rat, rat testis actin-like protein 3 (rTact3). The complete cDNA of the rTact3 gene was approximately 3.7 kb in length, and its corresponding amino acid sequence consisted of 1219 amino acids. The rTact3 gene lacks introns, similar to mTact1 and mTact2. The 356 C-terminal amino acids of rTact3 showed 43% homology with mTact1, whereas the 863 N-terminal amino acids did not show any significant homology. Northern blot analysis revealed that rTact3 mRNA was expressed only in adult rat testes and not during the prepubescent stage. In situ hybridization revealed that rTact3 was expressed exclusively during round and elongated spermatids maturation stages in rat testes. Immunohistochemical experiments using antibodies raised against a synthetic peptide showed that the expression of the rTact3 protein was also restricted in round and elongated spermatids, specifically in the head and acrosome of mature rat sperm. The 5'-flanking region of the mTact3 gene was found to contain a TATA-box motif as well as two putative CREB/c-Jun and five C/EBP motifs. mTact3 promoter activity was enhanced in a dose-dependent manner by the transfection of CREB, c-Jun, or C/EBP in NIH3T3 cells. These results suggest that Tact3 proteins might play an important role in rodent germ-cell development.

Anti-steroidogenic factor ARR19 inhibits testicular steroidogenesis through the suppression of Nur77 transactivation.

ARR19 (androgen receptor corepressor-19 kDa), a leucine-rich protein whose expression is down-regulated by luteinizing hormone and cAMP, is differentially expressed during the development of Leydig cells and inhibits testicular steroidogenesis by reducing the expression of steroidogenic enzymes. However, the molecular events behind the suppression of testicular steroidogenesis are unknown. In the present study, we demonstrate that ARR19 inhibits the transactivation of orphan nuclear receptor Nur77, which is one of the major transcription factors that regulate the expression of steroidogenic enzyme genes in Leydig cells. ARR19 physically interacts with Nur77 and suppresses Nur77-induced promoter activity of steroidogenic enzyme genes including StAR, P450c17, and 3beta-HSD in Leydig cells. Transient transfection and chromatin immunoprecipitation assays revealed that ARR19-mediated reduced expression of steroidogenic enzyme genes was likely due to the interference of SRC-1 recruitment to Nur77 protein on the promoter of steroidogenic enzyme genes. These findings suggest that ARR19 acts as a novel coregulator of Nur77, in turn regulating Nur77-induced testicular steroidogenesis, and may play an important role in the development and function of testicular Leydig cells.

Luteinizing hormone-stimulated pituitary adenylate cyclase-activating polypeptide system and its role in progesterone production in human luteinized granulosa cells.

The present study examined the gonadotropin regulation of pituitary adenylate cyclase-activating polypeptide (PACAP) and PACAP type I receptor (PAC(1)-R) expression, and its role in progesterone production in the human luteinized granulosa cells. The stimulation of both PACAP and PAC(1)-R mRNA levels by LH was detected using a competitive reverse transcription-polymerase chain reaction (RT-PCR). PACAP transcript was stimulated by LH reaching maximum levels at 12 hours in a dose dependent manner. LH treatment also stimulated PAC(1)-R mRNA levels within 24 hours. Addition of PACAP-38 (10(-7) M) as well as LH significantly stimulated progesterone production during 48 hours culture. Furthermore, co-treatment with PACAP antagonist partially inhibited LH-stimulated progesterone production. Treatment with vasoactive intestinal peptide, however, did not affect progesterone production. Taken together, the present study demonstrates that LH causes a transient stimulation of PACAP and PAC(1)-R expression and that PACAP stimulates progesterone production in the human luteinized granulosa cells, suggesting a possible role of PACAP as a local ovarian regulator in luteinization.

Attenuation of cell cycle progression by 2,3,7,8-tetrachlorodibenzo-p-dioxin eliciting ovulatory blockade in gonadotropin-primed immature rats.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) reduces ovulation rate in rats. The present study was to investigate whether TCDD alters the progression of cell cycle, and thus resulting in the blockade of ovulation in gonadotropin-primed, immature rats. The ovulation rate and ovarian weight were reduced in intact rats given TCDD (32 µg/kg BW in corn oil) by gavage one day before pregnant mare's serum gonadotropin (PMSG; 5 IU/rat) injection. Flow cytometry demonstrated that the percentage of granulosa cells in S-phase was increased at 24 h following PMSG treatment, but declined at 8 h following hCG treatment in corn oil-treated rats. Interestingly, the number of S-phase cells in TCDD-treated rats was reduced 24 and 48 h following PMSG treatment. TCDD, however, increased the percentage of cells in G2/M-phase at 24 h following PMSG treatment. TCDD inhibited the mRNA levels of Cdk2 at 0 h and 24 h, and cyclin D2 at 24 h and 48 h following PMSG treatment. Protein levels of aryl hydrocarbon receptor in granulosa cells were elevated in TCDD-treated rats at 12 h and 24 h following PMSG treatment. The present study indicates that TCDD reduces S-phase cells and inhibits levels of Cdk2 and cyclin D2 at 24 h following PMSG treatment, implying the ovulation-inhibiting action of TCDD may be exerted through the attenuation of cell cycle progression via AhR-mediated cascade.

The hedgehog-patched signaling pathway and function in the mammalian ovary: a novel role for hedgehog proteins in stimulating proliferation and steroidogenesis of theca cells.

The expression of hedgehog (Hh) genes, their receptor, and the co-receptor in mice, rat, and bovine ovaries were investigated. RT-PCR of ovarian transcripts in mice showed amplification of transcripts for Indian (Ihh) and desert (Dhh) Hh, patched 1 (Ptch1), and smoothened (Smo) genes. Semi-quantitative RT-PCR and northern blot analyses showed that whole ovarian Ihh and Dhh transcripts decreased 4-24 h after hCG versus 0-48 h after pregnant mares serum gonadotrophin treatment in mice, whereas mouse Ptch1 and Smo transcripts were expressed throughout the gonadotropin treatments. Quantitative real-time RT-PCR (qRT-PCR) revealed that the expression of the Hh-patched signaling system with Ihh mRNA abundance in granulosa cells was greater, whereas Smo and Ptch1 mRNA abundance was less in theca cells of small versus large follicles of cattle. In cultured rat and bovine theca-interstitial cells, qRT-PCR analyses revealed that the abundance of Gli1 and Ptch1 mRNAs were increased (P<0.05) with sonic hedgehog (SHH) treatment. Additional studies using cultured bovine theca cells indicated that SHH induces proliferation and androstenedione production. IGF1 decreased Ihh mRNA abundance in bovine granulosa cells. The expression and regulation of Ihh transcripts in granulosa cells and Ptch1 mRNA in theca cells suggest a potential paracrine role of this system in bovine follicular development. This study illustrates for the first time Hh activation of Gli1 transcriptional factor in theca cells and its stimulation of theca cell proliferation and androgen biosynthesis.

Hormonal regulation of proprotein convertase subtilisin/kexin type 5 expression during ovarian follicle development in the rat.

The proprotein convertase subtilisin/kexin (PCSKs), a family of subtilisin-like proteases, is the processing enzymes for the activation of many hormone precursors. The present study was designed to identify the PCSK isoform expressed in the ovary and to examine its expression in gonadotropin-stimulated rat ovary. Northern blot analysis of ovaries obtained from prepubertal rats revealed an increased expression of Pcsk5 messenger RNA (mRNA) during development with the highest levels at 21 days of age. Treatment of immature rats with PMSG further increased ovarian Pcsk5 expression, and in situ hybridization analysis revealed the localization of Pcsk5 mRNA in theca-interstitial cells of follicles in different sizes. Interestingly, treatment of PMSG-primed rats with hCG resulted in a transient stimulation of ovarian Pcsk5 mRNA levels within 3-6 h. In addition to theca-interstitial cells, hCG treatment induced the expression of Pcsk5 in granulosa cells of preovulatory follicles. Pcsk1, 2 and 4 mRNAs were not detected whereas Pcsk7 mRNA was slightly expressed. Injection of a progestin antagonist RU486 or an inhibitor of 3beta-hydroxysteroid dehydrogenase epostane at 1h before hCG treatment inhibited hCG-induced Pcsk5 mRNA levels. Treatment with LH stimulated both Pcsk5 mRNA and protein levels in preovulatory follicles cultured in vitro. In addition, forskolin but not TPA stimulated Pcsk5 mRNA levels. RNase protection assay revealed that the soluble Pcsk5A variant was the predominant form stimulated by gonadotropins in the ovary. Finally, the predicted proprotein substrates cleaved by PCSK5 were analyzed in preovulatory follicles using regular expressions. The present study demonstrates PCSK5A as the gonadotropin-regulated PCSK isoform in the ovary, and its possible contribution to ovulation by processing pro-TGFbeta and matrix metalloproteinase family.

Interferon-alpha enhances artemisinin-induced differentiation of HL-60 leukemia cells via a PKC alpha/ERK pathway.

Interferon-alpha (IFN-alpha) has been used in the treatment of several cancers, including chronic myeloid leukemia. Artemisinin, a sesquiterpene lactone endoperoxide that exists in several medicinal plants, is a well known anti-malarial agent. We previously reported that artemisinin by itself caused a relatively low level of HL-60 cell differentiation. In this study, we investigated the effects of IFN-alpha in combination with artemisinin on cell growth and differentiation in HL-60 leukemia cells. Combination of IFN-alpha and artemisinin synergistically induced the levels of leukemia cell differentiation, although IFN-alpha by itself did not affect cell proliferation and differentiation. The increased cell differentiation by IFN-alpha and artemisinin was significantly suppressed by the inhibitors for protein kinase C (PKC), extracellular signal-regulated kinase (ERK) and jun N-terminal kinase (JNK), but not by the inhibitors for phosphatidylinositol 3-kinase (PI3-K) and p38 mitogen-activated protein kinase (MAPK). Furthermore, co-treatment with IFN-alpha increased levels of PKC alpha and phosphorylated ERK. Taken together, these results indicate the enhancement of artemisinin-induced HL-60 cell differentiation by IFN-alpha through the activation of a PKC alpha/ERK signaling pathway, and suggest a possible use of IFN-alpha and artemisinin in the treatment of leukemic diseases.

Activation of protein kinase Czeta mediates luteinizing hormone- or forskolin-induced NGFI-B expression in preovulatory granulosa cells of rat ovary.

We have previously demonstrated that luteinizing hormone (LH) induces a rapid and transient expression of NGFI-B in the ovary. In this report, we investigated the signaling pathway for LH- and forskolin-induced NGFI-B expression in cultured rat granulosa cells of preovulatory follicles. LH- or forskolin-induced NGFI-B expression was suppressed by high dose of protein kinase C (PKC) inhibitor RO 31-8220 (10 microM), but not by low doses RO 31-8220 (0.1-1.0 microM) or adenylate cyclase inhibitor MDL-12,300A, implicating the involvement of atypical PKCs. Kinase assay revealed that LH treatment of granulosa cells resulted in a rapid stimulation of atypical PKCzeta activity. Interestingly, like LH, forskolin was also able to activate PKCzeta. Treatment with the cell-permeable PKCzeta-specific inhibitor pseudosubstrate peptide inhibited LH-or forskolin-induced NGFI-B expression, indicating the essential role of PKCzeta. Consistent with this promise, in granulosa cells depleted of diacylglycerol sensitive PKCs by prolonged treatment with tetradecanoylphobol-13-acetate, LH or forskolin could still induce NGFI-B expression, and RO 31-8220 or the PKCzeta pseudosubstrate peptide inhibited LH- or forskolin-induced NGFI-B expression. Furthermore, overexpression of dominant-negative PKCzeta in primary granulosa cells using a replication-defective adenovirus vector resulted in the suppression of LH- or forskolin-induced NGFI-B expression. Our findings demonstrate that PKCzeta, which is activated by LH or forskolin, contributes to the induction of NGFI-B in granulosa cells of preovulatory follicles.

Gonadotropin regulation of RIP140 messenger ribonucleic acid expression in the rat ovary.

Female mice null for receptor-interacting protein 140 (RIP140) are infertile because of the failure of follicle rupture. The present study examined gonadotropin regulation of RIP140 expression in immature rat ovary. Treatment with PMSG increased ovarian RIP140 mRNA and protein levels. In contrast, hCG treatment rapidly inhibited RIP140 mRNA and protein levels within 1-3 h. RIP140 mRNA was detected in theca cells of growing follicles in untreated ovary and in granulosa cells in PMSG-treated ovary. Interestingly, hCG treatment reduced RIP140 mRNA levels in granulosa cells of preovulatory follicles, but not of growing follicles. Neither treatment of immature rats with diethylstilbestrol in vivo nor of immature granulosa cells with FSH in vitro affected RIP140 mRNA levels. Treatment of immature granulosa cells with 17beta-estradiol in vitro, however, stimulated RIP140 mRNA levels. In cultured preovulatory granulosa cells, RIP140 mRNA levels were stimulated at 1 h and then declined to below control levels by 3 h after LH treatment. Treatment with MDL-12,330A, an inhibitor of adenylate cyclase, or chelerythrine chloride, an inhibitor of protein kinase C (PKC), inhibited LH-stimulated RIP140 gene expression. Furthermore, forskolin or TPA treatment for 1 h mimicked the stimulatory action of LH, indicating the involvement of both adenylate cyclase and PKC pathways. These results demonstrate the stimulation by PMSG and inhibition by hCG of RIP140 expression in granulosa cells of preovulatory follicles in the rat ovary.

Orchiectomy reduces hepatotumorigenesis of H-ras12V transgenic mice via the MAPK pathway.

A common characteristic of hepatocellular carcinoma in humans and animals is a striking male prevalence. However, the underlying mechanisms remain largely unknown. We have reported that hepatotumorigenesis is prevalent in male H-ras12V transgenic mice (Wang et al., 2005). Orchiectomy and ovariectomy were performed to determine if the presence of sex organ-related factors contributes to hepatotumorigenesis. After orchiectomy and ovariectomy, the mice were sampled at 6 months of age. The pathological diagnosis, sex hormone levels, expression of the H-ras12V transgene, and ras related signal pathways were determined. Orchiectomy significantly reduced the incidence of hepatotumorigenesis in males. However, no significant difference was detected in the incidence of tumorigenesis between ovariectomized and non-ovariectomized females. Molecular biochemical experiments showed that the sex organ-related factors significantly influenced transgene expression, which contributed to activation of the MAPK signaling pathway. The present study demonstrates that testis-related factors play important roles in hepatotumorigenesis in H-ras12V transgenic mice.

Phosphodiesterase regulation is critical for the differentiation and pattern of gene expression in granulosa cells of the ovarian follicle.

Feedback regulations are integral components of the cAMP signaling required for most cellular processes, including gene expression and cell differentiation. Here, we provide evidence that one of these feedback regulations involving the cyclic nucleotide phosphodiesterase PDE4D plays a critical role in cAMP signaling during the differentiation of granulosa cells of the ovarian follicle. Gonadotropins induce PDE4D mRNA and increase the cAMP hydrolyzing activity in granulosa cells, demonstrating that a feedback regulation of cAMP is operating in granulosa cells in vivo. Inactivation of the PDE4D by homologous recombination is associated with an altered pattern of cAMP accumulation induced by the gonadotropin LH/human chorionic gonadotropin (hCG), impaired female fertility, and a markedly decreased ovulation rate. In spite of a disruption of the cAMP response, LH/hCG induced P450 side chain cleavage expression and steroidogenesis in a manner similar to wild-type controls. Morphological examination of the ovary of PDE4D-/- mice indicated luteinization of antral follicles with entrapped oocytes. Consistent with the morphological finding of unruptured follicles, LH/hCG induction of genes involved in ovulation, including cyclooxygenase-2, progesterone receptor, and the downstream genes, is markedly decreased in the PDE4D-/- ovaries. These data demonstrate that PDE4D regulation plays a critical role in gonadotropin mechanism of action and suggest that the intensity and duration of the cAMP signal defines the pattern of gene expression during the differentiation of granulosa cells.