**2.2 Experimental design**

To study changes in proestrous LH and P surge characteristics with age, 4-month-old ('young') and 8.5-month-old ('middle-aged') female rats with regular 4-day cycles were used for blood sampling and hormone analyses. Hourly blood samples were taken on proestrus to measure plasma LH and P profiles. To investigate pituitary LH and FSH responsiveness, a potent GnRH analog (Ovalyse; des-Gly10-GnRH-ethylamide, Upjohn, Ede, The Netherlands) was used on the following proestrus. Ovalyse (100 ng in 0.25 ml 0.9% NaCl (w/v) containing 1% BSA) was administered i.v. immediately after the first blood sample was drawn.

Subsequently, a group of cycling females was ovariectomized (OVX, Van der Beek et al, 1999) at 4.5 (young) or 9 mo (middle-aged) of age, and given a single s.c. injection with estrogen at ZT3 on day 13 following OVX. Animals were perfused 2, 8, 26 or 32 hrs later, i.e. at ZT5 and ZT 11. Brains sections were stained for SOM peptide as described previously (Van Vugt et al, 2008).
