The LH surge triggers dramatic transcriptional changes in genes associated with ovulation and luteinization. The present study investigated the spatiotemporal expression of nuclear factor IL-3 (NFIL3), a transcriptional regulator of the basic leucine zipper transcription factor superfamily, and its potential role in the ovary during the periovulatory period. Immature female rats were injected with pregnant mare's serum gonadotropin, treated with human chorionic gonadotropin (hCG), and ovaries or granulosa cells were collected at various times after hCG. Nfil3 mRNA was highly induced both in intact ovaries and granulosa cells after hCG treatment. In situ hybridization demonstrated that Nfil3 mRNA was highly induced in theca-interstitial cells at 4-8 h after hCG, localized to granulosa cells at 12 h, and decreased at 24 h. Overexpression of NFIL3 in granulosa cells inhibited the induction of prostaglandin-endoperoxide synthase 2 (Ptgs2), progesterone receptor (Pgr), epiregulin (Ereg), and amphiregulin (Areg) and down-regulated levels of prostaglandin E2. The inhibitory effect on Ptgs2 induction was reversed by NFIL3 small interfering RNA treatment. In theca-interstitial cells the expression of hydroxyprostaglandin dehydrogenase 15-(nicotinamide adenine dinucleotide) (Hpgd) was also inhibited by NFIL3 overexpression. Data from luciferase assays demonstrated that NFIL3 overexpression decreased the induction of the Ptgs2 and Areg promoter activity. EMSA and chromatin immunoprecipitation analyses indicated that NFIL3 binds to the promoter region containing the DNA-binding sites of cAMP response element binding protein and CCAAT enhancer binding protein-β. In summary, hCG induction of NFIL3 expression may modulate the process of ovulation and theca-interstitial and granulosa cell differentiation by regulating expression of PTGS2, PGR, AREG, EREG, and HPGD, potentially through interactions with cAMP response element binding protein and CCAAT enhancer binding protein-β on their target gene promoters.
"For in vitro studies, animals were scarified at 48 h after PMSG administration and ovaries were collected to isolate and culture granulosa cells as described previously ; . Cells were cultured in HyQ MEM-RS (ThermoFisher Scientific, Waltham, MA, USA) media supplemented with 0.05 mg/ml gentamicin and 1× insulin, transferrin, and selenium, as well as with 1 I U/ml hCG to mimic LH action that induces ovarian gene expression and prostaglandins production in vitro
; . Cells were exposed to o,p’-DDT at concentration of 10−12 to 10−8 M for 6 h. "
[Show abstract][Hide abstract] ABSTRACT: o,p'-DDT is an infamous xenoestrogen as well as a ubiquitous and persistent pollutant. Biomonitoring studies show that women have been internally exposed to o,p'-DDT at range of 0.3-500 ng/g (8.46×10(-10) M-1.41×10(-6) M) in blood and other tissues. However, very limited studies have investigated the biological effects and mechanism(s) of o,p'-DDT at levels equal to or lower than current exposure levels in human. In this study, using primary cultures of rat ovarian granulosa cells, we determined that very low doses of o,p'-DDT (10(-12)-10(-8) M) suppressed the expression of ovarian genes and production of prostaglandin E2 (PGE2). In vivo experiments consistently demonstrated that o,p'-DDT at 0.5-1 mg/kg inhibited the gene expression and PGE2 levels in rat ovary. The surprising results from the receptor inhibitors studies showed that these inhibitory effects were exerted independently of either classical estrogen receptors (ERs) or G protein-coupled receptor 30 (GPR30). Instead, o,p'-DDT altered gene expression or hormone action via inhibiting the activation of protein kinase A (PKA), rather than protein kinase C (PKC). We further revealed that o,p'-DDT directly interfered with the PKA catalytic subunit. Our novel findings support the hypothesis that exposure to low concentrations of o,p'-DDT alters gene expression and hormone synthesis through signaling mediators beyond receptor binding, and imply that the current exposure levels of o,p'-DDT observed in the population likely poses a health risk to female reproduction.
PLoS ONE 11/2012; 7(11):e49916. DOI:10.1371/journal.pone.0049916 · 3.23 Impact Factor
"The AdEasy XL adenoviral vector system kit (Stratagene) was used to construct Plk2 and Plk3 recombinant adenovirus. The processes for generating and propagating recombinant adenoviruses were described previously . Briefly, the full length of rat Plk2 and Plk3 genes were cloned into the pShuttle-CMV vector and then a recombinant Ad-Plk2 and Ad-Plk3 plasmids were generated by homologous recombination. "
[Show abstract][Hide abstract] ABSTRACT: The highly conserved polo-like kinases (Plks) are potent regulators of multiple functions in the cell cycle before and during mitotic cell division. We investigated the expression pattern of Plk genes and their potential role(s) in the rat ovary during the periovulatory period. Plk2 and Plk3 were highly induced both in intact ovaries and granulosa cells in vivo after treatment with the luteinizing hormone (LH) agonist, human chorionic gonadotropin (hCG). In vitro, hCG stimulated the expression of Plk2 in granulosa cells, but not Plk3. This induction of Plk2 expression was mimicked by both forskolin and phorbol 12 myristate 13-acetate (PMA). Moreover, Plk2 expression was reduced by inhibitors of prostaglandin synthesis or the EGF pathway, but not by progesterone receptor antagonist (RU486) treatment. At the promoter level, mutation of the Sp1 binding sequence abolished the transcriptional activity of the Plk2 gene. ChIP assays also revealed the interaction of endogenous Sp1 protein in the Plk2 promoter region. Functionally, the over-expression of Plk2 and Plk3 arrested granulosa cells at the G0/G1 phase of the cell cycle. In contrast, the knockdown of Plk2 expression in granulosa cells decreased the number of cells in the G0/G1 stage of the cell cycle, but increased granulosa cell viability. In summary, hCG induced Plk2 and Plk3 expression in the rat ovary. Prostaglandins and the EGF signaling pathway are involved in regulating Plk2 expression. The transcription factor Sp1 is important for Plk2 transcriptional up-regulation. Our findings suggest that the increase in Plk2 and Plk3 expression contributes to the cell cycle arrest of granulosa cells which is important for the luteinization of granulosa cells during the periovulatory period.
PLoS ONE 08/2012; 7(8):e41844. DOI:10.1371/journal.pone.0041844 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Bifenthrin (BF), a broad-spectrum and widely used synthetic pyrethroid, is a typical chiral pesticide. More attention is being paid to the health risk assessment of the enantioselective toxicity of BF isomers. In this study, we used rat ovarian granulosa cells as in vitro model to investigate effects of BF enantiomers on the biosynthesis of two hormones, progesterone and prostaglandin E2 (PGE2), which are critical for mammalian reproduction. We showed that 1S-cis-BF, but not 1R-cis-BF significantly decreased the secretion of progesterone and PGE2 in granulosa cells. 1S-isomer of BF reduced the expression of genes P450scc, StAR, PBR and DBI, as well as COX-2, which are involved in regulating the rate-limiting steps of progesterone or PGE2 biosynthesis. The transcriptional activation of StAR and COX-2 promoter were disrupted by 1S-cis-BF. Furthermore, activity of protein kinase C (PKC), an important signaling mediator of progesterone and PGE2 synthesis, was differentially inhibited by 1S-cis-BF. The data of molecular docking revealed that one hydrogen bond was formed between 1S-cis-BF and PKC protein. In conclusion, we firstly reported in this study the enantioselective disrupting effects of BF isomers on progesterone and PGE2 synthesis via PKC pathway in rat ovarian cells. Our findings suggest that the enantioselective toxicity of chiral pesticides should be considered for evaluating mammalian reproductive health, a toxicologic endpoint of great concern in health risk assessment.
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