Serum hormone analyses

The blood was allowed to clot for 15 minutes and then centrifuged for 10 minutes at 2500 rpm. The serum was collected and frozen at −80°F. Pituitaries were snap frozen. Serum was analyzed at the University of Virginia Ligand Core through enzyme-linked immunosorbent assay (ELISA) multiplex, in duplicate or using in-house FSH and LH assays, as previously described (28). Specifically for the FSH assay, we used a Luminex Assay (Millipore, Burlington, MA), which results in ∼10-fold lower FSH concentrations compared with the ELISA multiplex assay used by the University of Virginia Ligand Core. Whereas this is a consistent result, the reasons for this difference are unclear, as the Luminex Assay contains proprietary reagents. As a consequence, we considered relative changes in FSH concentrations. Testosterone was assayed using a commercially available kit (IBL, Minneapolis, MN), per the manufacturer’s instructions.