Genomics of estradiol-3-sulfate action in the ovine fetal hypothalamus

Abstract

In fetal sheep during late gestation sulfoconjugated estrogens in plasma reach a concentration 40 -100 times greater than unconjugated estrogens. The objective of the present study was to determine the genomics of estradiol-3-sulfate (E2S) action in the ovine fetal brain. The hypothesis was that E2S stimulates genes involved in the neuroendocrine pathways that direct or facilitate fetal development at the end of gestation. Four sets of chronically catheterized ovine twin fetuses were studied (gestational age: 120 -127 days gestation) with one infused with E 2S intracerebroventricularly (1 mg/day) and the other remaining untreated (control). After euthanasia, mRNA samples were extracted from fetal brains. Only hypothalamic samples were employed for this study given the important function of this brain region in the control of the hypothalamus-pituitary-adrenal axis. Microarray analysis was performed following the Agilent protocol for one-color 8 ϫ 15 microarrays, designed for Ovis aries. A total of 363 known genes were significantly upregulated by the E 2S treatment (P Ͻ 0.05). Network and enrichment analyses were performed using the Cytoscape/Bingo software, and the results validated by quantitative realtime PCR. The main overrepresented biological processes resulting from this analysis were feeding behavior, hypoxia response, and transforming growth factor signaling. Notably, the genes involved in the feeding behavior (neuropeptide Y and agouti-related protein) were the most strongly induced by the E2S treatment. In conclusion, E2S may be an important component of the mechanism for activating orexigenic, hypoxia responsiveness and neuroprotective pathways in the lamb as it approaches postnatal life. heart rate; estradiol; hypoxia; estrogen receptor; neuropeptides; neurosteroid IN FETAL SHEEP and pregnant ewes, sulfoconjugated estrogens are far more abundant than unconjugated estrogens (9,41,46). High concentrations of estrone sulfate in uterine vein plasma (compared to peripheral vein plasma) of pregnant sheep suggested a high secretion rate for this steroid by placenta in late gestation (15). Plasma concentrations of estradiol-3-sulfate (E 2 S) are ϳ40 -100 times those of estradiol (E 2 ) in the lategestation fetal sheep. E 2 S is taken up by the fetal brain and stimulates responses that are both similar to and distinct from the responses to E 2 (43, 46). Sulfoconjugation increases the half-life in the blood (33) and supplies a ready source of E 2 in tissues (e.g., hypothalamus) that express steroid sulfatase (STS) (31, 43). E 2 S can bind estrogen receptor only after deconjugation by STS. We have proposed that, while the function of the sulfoconjugated estrogens in fetal and maternal sheep is unknown, deconjugation can increase estrogen action in specific tissues that express STS and estrogen receptor (43,44,46). The fetal brain expresses both STS and estrogen sulfotransferase (STF), allowing for the bidirectional interconversion of E 2 and E 2 S (31, 32), although the ratio of expression for these enzymes fa...

Genomics of the fetal hypothalamic cellular response to transient hypoxia: endocrine, immune, and metabolic responses

“…To summarize the results of this gene array study, we have used the Agilent 15k ovine array, previously annotated in our laboratory (31), to identify genes in the ovine fetal hypothalamus that are significantly up-and downregulated 1 h after the onset of a 30 min period of maternal ventilatory hypoxia. There were major alterations in the transcriptome in response to the hypoxia.…”

“…The labeled cRNA was stored at Ϫ80°C until use. Microarray hybridization, washing, and scanning were performed as previously described (31).…”

“…We used functional annotation of the genes as a useful tool to categorize the genes into classes, as previously described (31). Also as in our previous study, the platform number is GPL14112 (Agilent-019921 Sheep Gene expression microarray 8 ϫ 15K, G4813A).…”

“…), as dictated by the individual hypotheses. We have recently annotated the Agilent 15k ovine array, and we have used it to measure changes in gene expression in fetal hypothalamus in response to infusion of estradiol-3-sulfate (31). In the present experiment, we have used this array to investigate the transcriptomics of the ovine fetal hypothalamic response to hypoxia, testing the hypothesis that the acute changes in the transcriptome in response to transient hypoxia can be described as a network of genes known to interact with each other.…”

Transcriptomics of the fetal hypothalamic response to brachiocephalic occlusion and estradiol treatment

“…The array platform that we used was the Agilent-019921 Sheep Gene Expression Microarray 8x15k, G4813A (GPL14112). Annotation of this array platform was previously described (23). Data have been deposited in the Gene Expression Omnibus database (accession number GSE52888).…”