**3.4 SOM-ir cells and fibers in the PeVN**

Total numbers of SOM-ir cells were roughly comparable between age groups (Figure 1). In the young animals, SOM-ir numbers were not significantly affected by time after E2 treatment, although they appeared to be consistently lower at ZT5 compared to ZT11 (Figure 1A). In middle-aged rats, total numbers of SOM-ir cells were significantly lower at ZT 5 on day 1 compared to day 2 (Figure 1B).

SOM-ir cells within the PeVN showed a clear rostro-caudal distribution pattern, with maximal numbers of cells appearing in the more caudal part of the PeVN. The distribution in young females varied slightly over the different time points after E2 treatment: maximal numbers of SOM-ir cells were found consistently in PeVN section 8 at ZT5, but in PeVN section 7 at ZT11 on both days (Figure 2A and B). In middle aged female, the rostro-caudal distribution pattern in the number of SOM-ir cells at ZT 5 on day 1 (Figure 2C) was absent, e.g. the number of SOM-ir cells was comparable between PeVN sections. Distribution

Plasma P levels increased gradually during proestrus, but did not show a distinct peak during the time window evaluated. Basal P levels defined by LH release characteristics showed no significant differences between groups and age did not affect P profiles (Table 1). Total P levels correlated significantly with basal P levels (r=0.876 with *P* < 0.001) and with P surge levels (r=0.992 with *P* < 0.001), in line with the observed elevation in P levels during

Administration of Ovalyse at ZT 5.5 resulted in a rapid and consistent increase in LH plasma levels irrespective of age (Table 2). Highest plasma LH levels were measured at 1 or 2 h after Ovalyse administration and decreased thereafter (defined as the 'early', induced LH surge). After ZT 8.5, LH levels increased again (defined as the 'late', endogenous LH

LH levels were of comparable magnitude at 1 and 2 h after Ovalyse injection between 4 and 8.5 months old females. Also, the second, endogenous LH surge was comparable in magnitude (peak height and total LH levels) between groups and accompanied by a gradual

peak height total peak height total total total total total 4 12 30.0 ± 1.6 66.9 ± 2.5 14.8 ± 1.7 48.9 ± 5.4 115.9 ± 6.0 172.9 ± 12.2 365.5 ± 30.3 538.3 ± 42.0 8.5 10 31.0 ± 3.1 63.4 ± 7.2 16.3 ± 2.2 55.0 ± 7.7 118.4 ± 12.6 157.8 ± 17.9 338.2 ± 45.6 496.0 ± 61.9

all samples <ZT9 >ZT9 all samples

P following Ovalyse®

Table 2. LH and P surge characteristics following Ovalyse® administration in young and middle-aged 4-day cyclic rats on proestrus. The surge was divided into a 'early' part (ZT<9; 'induced' surge) and a 'late' surge (ZT>9; 'endogenous' surge). Measured characteristics: peak height of the 'early' and 'late' LH surge (the highest concentration measured), and the total amount of LH or P released during the 'early' and 'late' and the entire sampling period (cumulative LH or P levels during the corresponding sampling periods). All data are

Total numbers of SOM-ir cells were roughly comparable between age groups (Figure 1). In the young animals, SOM-ir numbers were not significantly affected by time after E2 treatment, although they appeared to be consistently lower at ZT5 compared to ZT11 (Figure 1A). In middle-aged rats, total numbers of SOM-ir cells were significantly lower at

SOM-ir cells within the PeVN showed a clear rostro-caudal distribution pattern, with maximal numbers of cells appearing in the more caudal part of the PeVN. The distribution in young females varied slightly over the different time points after E2 treatment: maximal numbers of SOM-ir cells were found consistently in PeVN section 8 at ZT5, but in PeVN section 7 at ZT11 on both days (Figure 2A and B). In middle aged female, the rostro-caudal distribution pattern in the number of SOM-ir cells at ZT 5 on day 1 (Figure 2C) was absent, e.g. the number of SOM-ir cells was comparable between PeVN sections. Distribution

**3.2 Proestrous P levels** 

the entire sampling period.

surge).

(mo) <sup>n</sup>

**3.3 Pituitary responsiveness** 

increase in P levels comparable between ages.

<ZT9 >ZT9 age

expressed as group means SEM in ng/ml (concentrations).

LH following Ovalyse®

**3.4 SOM-ir cells and fibers in the PeVN** 

ZT 5 on day 1 compared to day 2 (Figure 1B).

patterns at other time points were in general comparable with those found in the young rats, i.e. maximal numbers of SOM-ir cells in PeVN section 7 at ZT 11 and in PeVN section 8 at ZT 5 on day 2 (Figure 2D).

Fig. 1. Total number of SOM-ir cells (sum of PeVN sections 4-8) in young (4.5 months old) and middle aged (9 mo old) Wistar rats at different time points after E2 treatment. ZT 5: 2 (day 1) or 26 (day 2) h after E2 treatment, ZT 11: 8 (day 1) or 32 (day 2) h after E2 treatment. n=5 for each young age group, numbers within base of bars indicate the number of animals.

Fig. 2. Rostral to caudal distribution of SOM-ir cells in the PeVN of young (4.5 months old) (A and B) and middle aged (C and D) OVX females at different time points after E2 treatment. Numbers within base of bars indicate the number of animals.

Somatostatin in the Periventricular Nucleus of the Female Rat:

GnRH priming mechanism may be particularly affected.

**4.2 Reproductive aging and the ovary** 

1984) and impaired GnRH priming (Brito et al,1994; Keizer et al, 2001).

explained by an increased responsiveness to LH stimulation.

**4.3 Reproductive aging and the hypothalamus** 

**4.1 Reproductive aging and the pituitary gland** 

Age Specific Effects of Estrogen and Onset of Reproductive Aging 79

The attenuation of the natural LH surge at 8.5-months old is in accordance with previous reports concerning other rat strains (Brito et al, 1984; DePaolo et al, 1986; Krieg et al, 1995; Nass et al, 1984). Some studies suggested that the decrease in proestrus LH levels with age may follow changes at the level of the pituitary gland, such as changes in LH storage and/or release capacity (Matt et al, 1998; Wise et al, 1984). The results of the present study, however, suggest that this is not the case. Although the timing of GnRH analog administration was early (i.e. 3 to 4 hours before the natural LH surge occurred), no age-related differences in total and peak LH levels of the 'induced' LH surge (until ZT9) were observed. This implies that LH responsiveness to a bolus of GnRH is comparable between 4- and 8.5-month-old rats. Others did show that the acutely releasable pool of LH was reduced at the age of 9-12 months in cyclic Sprague-Dawley rats (Brann and Mahesh, 2005; Wise et al, 1984). In addition, pituitary responsiveness to GnRH in vitro is decreased in 10-12 month-old Long-Evans rats that show attenuated LH surges (Brito et al, 1994), and in pituitaries from 9 compared to 4-month-old Wistar rats that were tested in a superfusion system in our lab (Keizer et al, 2001). Since the age-related reduction in LH release after GnRH stimulation was more evident during the second and third stimulus in all studies, this suggests that the

Yet, we found no age-related differences in total LH levels of the 'late', 'endogenous' LH surge that results from endogenous GnRH release. Since the LH surge requires repeated pulses of GnRH to induce full pituitary priming, the absence of these age-related changes in this study suggest that GnRH priming is not significantly affected in our 8.5-month-old rats. The time between GnRH stimuli, however, differs between endogenous GnRH release (~1 hour between pulses) and our stimulus with the long-acting GnRH-analog Ovalyse® (~3 hours). Altogether, these results indicate that in our 8.5-month-old females, the attenuation of the LH surge is not caused by a diminished responsiveness of LH to initial GnRH signaling, although reproductive aging may eventually result in a decrease in the releasable pool of LH (Wise et al,

In the present study we showed that proestrous P levels were comparable between 4- and 8.5-month-old rats, and thus do not underlie the observed attenuation of the LH surge. In contrast, another study (Miller and Riegle, 1980) showed that the attenuated preovulatory LH surge was accompanied by an attenuated P surge in 12-month-old cyclic Long-Evans rats. It has been suggested that attenuated P levels result from a decrease in proestrous LH levels, although increased responsiveness of the ovary to hCG stimulation in regular cyclic middle-aged compared has been reported for Long-Evans rats (Chern et al, 2000). Consequently, the lack of concurrent changes in P and LH release in our rats could be

Based on these data, we hypothesize that the initial attenuation of the LH surge is indeed initiated by alterations at the hypothalamic level (i.e. GnRH release), and not at the pituitary

A previous study by Rubin (Rubin, 1992) showed that the secretory capacity of the GnRH system is still intact in middle-aged rats, but that the LH secretion per GnRH burst during

gland (i.e. responsiveness to GnRH, GnRH priming) or the ovary (P levels).

In young animals, the area occupied by SOM-ir fibers in the PeVN region was significantly different at ZT5 on day 1 compared to ZT11 day 2 (Figure 3A). The total area of SOM-ir fibers, i.e. including the fibers projecting to the ME, was significantly different between ZT5 and ZT11 on both days (Figure 3B).

Fig. 3. Area of SOM-ir fibers in the PeVN region (A) or total SOM-ir area (B) in young (4.5 months old) OVX female Wistar rats at different time points after E2 treatment. a significantly different from b (p=0.047; Bonferroni); c significantly different from d (p ≤ 0.05; Tukey HSD). n=5 for each group.
