**2.4 Cannulation and blood sampling**

The right jugular vein of female rats was cannulated to obtain stress-free blood samples (Steffens, 1969, Van der Beek et al 1999, Van Vugt et al, 2004). After a recovery period of at least five days, ten hourly blood samples of 170 l were taken on proestrus from ZT 5.5-14.5 for measurement of endogenous, preovulatory hormone profiles. To assess the amount of acutely releasable LH by the pituitary gland, ten hourly blood samples of 170 l were drawn from ZT 5.5-14.5 on the following proestrus, following an i.v. injection of the GnRH analog Ovalyse, just after the first sample. Blood samples were collected in heparinized, air-dried

Somatostatin in the Periventricular Nucleus of the Female Rat:

**2.7 Statistics** 

hoc test.

(mo) <sup>n</sup>

**3. Results** 

**3.1 Proestrous LH profiles** 

LH surge age

Age Specific Effects of Estrogen and Onset of Reproductive Aging 75

showed a clear distinction between 'early', Ovalyse induced LH release and a second 'late' increase in LH levels resulting from endogenous proestrous GnRH release. Therefore, LH data after Ovalyse administration were divided in ZT 5.5-8.5 ('early') and ZT 9.5-14.5 ('late'). For 'late' release, LH peak time and LH peak height were determined as described above. Finally, total LH levels during the complete sampling period were calculated. As proestrous P release continuously increased but did not peak in the time window evaluated,

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

The general profile of the LH surge was comparable between ages. Basal plasma LH levels averaged 0.3 to 0.4 ng/ml and onset of the surge occurred around ZT9.5, where after LH levels increased rapidly and reached peak levels around ZT12. Subsequently, LH levels gradually declined. We found a significant decrease in LH peak and total LH levels with age (*P*=0.041 and *P* = 0.035, respectively) (Table 1). Pearson correlation tests showed that the magnitude of the LH surge (i.e. total LH levels) correlated with onset and height of the LH surge (LH onset time: r=-0.527 with *P* < 0.001; LH peak levels: r=0.924 with *P* < 0.001).

Table 1. Proestrous LH and P surge characteristics of young and middle-aged 4-day cyclic WU rats. Basal levels are depicted as the average concentration ZT 5.5-7.5, onset time of the LH surge (ZT at which LH levels exceeded basal levels plus 3xSD), peak time of the LH surge (ZT at which the highest concentration was measured), peak height of the LH surge (highest concentration measured), preovulatory P surge levels (cumulative value from ZT 8.5-14.5), and the total amount of LH or P released during the surge (cumulative value during the complete sampling period). All data are expressed as group means SEM in ng/ml (concentrations) or h:min (time). Significant differences (*P* < 0.05) between young and

4 12 0.3 ± 0.1 9:43 ± 0:30 12:06 ± 0:20 12.8 ± 1.4 40.2 ± 4.5 24.9 ± 2.6 339.4 ± 28.8 414.2 ± 35.9 8.5 9 0.4 ± 0.1 9:37 ± 0:23 11:37 ± 0:19 8.3 ± 1.4\* 25.2 ± 4.6\* 23.2 ± 5.0 329.0 ± 64.6 398.6 ± 78.7

basal onset time peak time peak height total basal surge total

P surge

middle aged with ages are indicated with an asterisk.

only basal level, preovulatory surge level, and total amount released were assessed.

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 6.2% for P analysis.

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

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 Complexelite (ABC; Vector Laboratories).

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

#### **2.6 Data processing**

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 showed a clear distinction between 'early', Ovalyse induced LH release and a second 'late' increase in LH levels resulting from endogenous proestrous GnRH release. Therefore, LH data after Ovalyse administration were divided in ZT 5.5-8.5 ('early') and ZT 9.5-14.5 ('late'). For 'late' release, LH peak time and LH peak height were determined as described above. Finally, total LH levels during the complete sampling period were calculated. As proestrous P release continuously increased but did not peak in the time window evaluated, only basal level, preovulatory surge level, and total amount released were assessed.
