A Role for Nuclear Factor Interleukin-3 (NFIL3), a Critical Transcriptional Repressor, in Down-Regulation of Periovulatory Gene Expression

College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 310036, People's Republic of China.
Molecular Endocrinology (Impact Factor: 4.02). 03/2011; 25(3):445-59. DOI: 10.1210/me.2010-0250
Source: PubMed


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.

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    • "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 [29]; [30]. 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 [32]; [33]. Cells were exposed to o,p’-DDT at concentration of 10−12 to 10−8 M for 6 h. "
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    • "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 [17]. 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. "
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