**2.7 Statistics**

74 Sex Steroids

vials (25 IU heparin, Leo Pharma BV, Breda, NL) and centrifuged at 13,000 rpm for 5 minutes. Plasma was diluted 1:4 for LH and 1:20 for P analysis with PBS buffer (0.02M, pH 7.5) containing 0.1% BSA, and stored frozen at –20 C until RIA. LH and P plasma levels were determined by validated RIAs (Van der Beek et al, 1999, Van der Meulen et al, 1988). Only samples from animals that displayed regular 4-day estrous cycles were included in the analysis. The inter- and intra-assay coefficients of variation were determined using pooled rat serum, and amounted to respectively 12.1% and 10.8 % for the LH assay and 15.8% and

Forty-three regularly cycling female rats aged 4.5 (n=20) or 9 (n=23) months were ovariectomized and treated with estradiol benzoate before perfusion 2, 8, 26 or 32 hours later. The brains were processed for SOM immunocytochemistry as described in detail previously (Van der Beek et al, 1991; Van Vugt et al, 2008). Staining was performed in two separate runs (4.5 and 9 mo) and intra-assay variation was controlled for by including a group of young animals at 2 and 32 h after E2 treatment in the second run. Every third brain section containing the PeVN was stained for SOM peptide by free-floating immunocytochemistry techniques. For staining, primary polyclonal rabbit antibody raised against SOM peptide (Somaar 080289, NIN, Amsterdam, NL) (Buijs et al, 1989) was used followed by detection with biotinylated goat anti-rabbit IgG and Avidin-Biotin Complex-

SOM-immunoreactive (-ir) neurons in the PeVN of the left side of the brain were counted using computer assisted analysis as described previously (Van Vugt et al, 2008). In addition to counting SOM-ir cells, also the amount/quantity of SOM-ir fibers (expressed in µm2) was measured in these images in the young females only. To this end, both the fibers that were located closely to the SOM cells (the "PeVN region": measured in an area that had an absolute distance from the ventricle of approximately 200 µm) and all fibers that originated from SOM cells in the PeVN, including those projecting to the ME ("total fibers": measured in an area that had an absolute distance from the ventricle of approximately 560 µm) were counted. The analysis threshold was determined in a representative selection of the images by measuring the mean gray level in an area devoid of SOM staining. Next, an upper and a lower threshold were determined (mean gray level + 3x S.D.; mean maximal gray level – 3x

To determine the effects of age on the proestrus LH and P surge several profile characteristics were defined: i.e. basal levels, onset time, peak time, peak height and the total amount of LH and P released. Basal levels were defined as the average concentration of the first three blood samples (ZT 5.5, 6.5, and 7.5) per animal. In case of an early rise in LH levels, i.e. at ZT 7.5 (n=3), the first two blood samples were used to calculate basal levels. Onset time was defined as the sample hour (ZT; mean SEM expressed as h:min min) at which LH levels exceeded basal LH levels plus 3 x the standard deviation, while LH levels continued to rise thereafter. Peak time of the LH surge was defined as the ZT hour at which the highest LH concentration was measured. The highest amount of LH measured at that time was defined as the peak height. The total amount of LH or P was defined by the cumulative value of hormone levels during the complete sampling period. LH levels

**2.5 Tissue processing, SOM immunocytochemistry & analysis** 

S.D. respectively) excluding SOM-ir cells and very light SOM-ir fibers.

6.2% for P analysis.

elite (ABC; Vector Laboratories).

**2.6 Data processing** 

Hormone levels were expressed as mean SEM and analyzed using SPSS (version 12.0). Differences were considered to be significant when P<0.05. Basal LH levels, onset time and peak time of the LH surge, LH peak height, and basal P levels from proestrus measurements, as well as LH peak height and total LH levels following Ovalyse® were tested with the nonparametric Kruskal-Wallis test and were post-hoc tested using the Mann-Whitney test. Changes with age in the total amount of LH and P released during the proestrous surge, preovulatory P surge levels as well as LH peak height of the induced LH surge and total LH levels following Ovalyse®) were tested by one-way ANOVA. To compare the total number of SOM-ir cells between the different time points following E2 treatment, one-way ANOVAs were used. A Bonferroni or Tukey HSD test was used as post hoc test.
