Enhanced at puberty 1 (EAP1) is a new transcriptional regulator of the female neuroendocrine reproductive axis
Sabine Heger, … , Wolfgang Sippell, Sergio R. Ojeda
Sabine Heger, … , Wolfgang Sippell, Sergio R. Ojeda
Published August 1, 2007
Citation Information: J Clin Invest. 2007;117(8):2145-2154. https://doi.org/10.1172/JCI31752.
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Enhanced at puberty 1 (EAP1) is a new transcriptional regulator of the female neuroendocrine reproductive axis

Abstract

The initiation of mammalian puberty and the maintenance of female reproductive cycles are events controlled by hypothalamic neurons that secrete the decapeptide gonadotropin-releasing hormone (GnRH). GnRH secretion is, in turn, controlled by changes in neuronal and glial inputs to GnRH-producing neurons. The hierarchical control of the process is unknown, but it requires coordinated regulation of these cell-cell interactions. Here we report the functional characterization of a gene (termed enhanced at puberty 1 [EAP1]) that appears to act as an upstream transcriptional regulator of neuronal networks controlling female reproductive function. EAP1 expression increased selectively at puberty in both the nonhuman primate and rodent hypothalamus. EAP1 encoded a nuclear protein expressed in neurons involved in the inhibitory and facilitatory control of reproduction. EAP1 transactivated genes required for reproductive function, such as GNRH1, and repressed inhibitory genes, such as preproenkephalin. It contained a RING finger domain of the C3HC4 subclass required for this dual transcriptional activity. Inhibition of EAP1 expression, targeted to the rodent hypothalamus via lentivirus-mediated delivery of EAP1 siRNAs, delayed puberty, disrupted estrous cyclicity, and resulted in ovarian abnormalities. These results suggest that EAP1 is a transcriptional regulator that, acting within the neuroendocrine brain, contributes to controlling female reproductive function.

Authors

Sabine Heger, Claudio Mastronardi, Gregory A. Dissen, Alejandro Lomniczi, Ricardo Cabrera, Christian L. Roth, Heike Jung, Francesco Galimi, Wolfgang Sippell, Sergio R. Ojeda

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Figure 4

Lentivirus-mediated delivery of EAP1 siRNAs decreases EAP1 mRNA expression both in vitro and in vivo.

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Lentivirus-mediated delivery of EAP1 siRNAs decreases EAP1 mRNA expressi...
(A) Diagram of the lentivirus construct (27) used, showing the site of insertion of a U6 promoter–driven shRNA-expressing cassette. (B) Generation of EAP1 sh1 from the U6 promoter–directed transcriptional cassette. (C) Inhibition of EAP1 gene expression in HiB5 hippocampal neural progenitor cells by EAP1 sh1 and -3, measured 48 hours after infection. EAP1 mRNA content was detected by semiquantitative PCR. Bars are mean ± SEM; n = 6–7 wells/group, except in the case of noninfected control (n = 3). *P < 0.05; ***P < 0.01 versus control or LV-infected group. (D and E) Lack of changes in 2ι,5ι oligoadenylate synthetase–1 (OAS1) mRNA abundance after in vitro infection of hypothalamic slices with lentiviral particles carrying EAP1 sh1. The slices were prepared and infected as described in Supplemental Note 7. OAS1 mRNA was measured by semiquantitative PCR 72 hours after infection. (D) PCR products size-fractionated in a 2% agarose gel stained with ethidium bromide. –RT, no reverse transcription product. MM, molecular markers. (E) Densitometric analysis of the gel shown in D. Cyclophilin (Cyclo) mRNA, a housekeeping gene, was used as the normalizing unit. Accordingly, OAS1 mRNA levels are expressed as mean ± SEM (AU) of the OAS1/cyclophilin mRNA ratio calculated for each group (n = 3).