Compelling evidence suggests that ovarian estrogen (E) indirectly regulates hypothalamic gonadotropin-releasing hormone (GnRH) secretion since E receptors are not found in GnRH neurons. The unique neural norepinephrine (NE) system and its well established ability to stimulate GnRH secretion has led us to hypothesize that E acts on NE-producing neurons whose cell bodies are located in the brainstem and whose neural endings coexist with hypothalamic GnRH neurons. To address this issue, we utilized the in situ hybridization (ISH) method to compare tyrosine hydroxylase (TH, the rate-limiting enzyme for NE synthesis) gene expression in the brainstem of OVX (n=5) and E-treated OVX monkeys. Two levels of E were given one group received E through capsules that maintained blood levels between 50-150 pg E/ml plasma (n=5); the second group received a similar E implant plus a s.c. injection of estradiol benzoate (EB, 42 fg/kg BW) that produced circulating values of >300 pg E/ml for several h (n=5). In preliminary trials, the supplemental E/EB treatment induced plasma LH surges between 24 and 48 h after EB injection. For ISH, the brains were perfused with 4% paraformaldehyde in 3.8% borate (pH=9.5). The 5 EB-treated brains were perfused at 30 h post-EB. The 15 brainstems were sectioned at 20 fm thick and the sections containing NE cells, i.e., locus coeruleus (LC) were labelled with a S-35 macaque specific TH cRNA fragment (176 bp cloned by us). The optical density of hybridized and nonspecific signals at the same level of the LC from each animal was determined from Cornex X-ray films illuminated by a ChromaPro 45 light source. Images were captured with a Dage MT1 70 video camera and digitized images were analyzed with NIH Image software. The net optical density (after background subtraction) was determined for each animal. This averaged density was used as an index of TH mRNA expression for each treatment group. The results suggest that TH transcription in the LC is upregulated by E, particularly after EB treatment. Since E binding occurs in several areas of the brainstem, including the LC, we propose that E-initiated ovulation exemplifies a brainstem-hypothalamic-pituitary-ovarian axis in primates. The extension of this axis to include the brainstem could open new research avenues for improving reproduction-associated health problems.
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