**4.1 Reproductive aging and the pituitary gland**

78 Sex Steroids

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

Fig. 3. Area of SOM-ir fibers in the PeVN region (A) or total SOM-ir area (B) in young (4.5

significantly different from b (p=0.047; Bonferroni); c significantly different from d (p ≤ 0.05;

In the present study we showed that in adult female rats, the effects of E2 on SOM-ir cell distribution and SOM-ir numbers in the PeVN were age dependent. Estrogen did not affect total numbers of SOM-ir cells in the PeVN of young female rats in line with our previous studies (Van Vugt et al, 2008) and those of others (Estupina et al, 1996). Other studies reported a decrease in SOM mRNA content in the PeVN following OVX which was reversed by E2 treatment (Baldino et al, 1988; Zorilla et al, 1991). In these studies, however, animals were treated with E2 for a prolonged period of time, whereas we studied the effect of a single physiological dose of E2 on SOM peptide-containing cells on multiple time points following the estrogen exposure. Interestingly, the amount of SOM-ir fibers within the PeVN region of young females was decreased at 32 h compared to 2 h after E2, which may suggest increased release of SOM peptide from the PeVN or decreased transport from cells to the fibers, apparently without affecting the amount of SOM peptide synthesized and/or stored in the PeVN cells. Prior to measuring SOM peptide responsiveness to an estrogen stimulus we showed that the attenuation of the LH surge at middle-age was not accompanied by a decrease in proestrus P levels or a decrease in pituitary LH responsiveness to a GnRH analog. These results clearly suggest that the attenuation of the LH surge is not initiated by alterations at the level of the ovary or pituitary gland, but rather the result of changes in response to ovarian feedback at the hypothalamic level as found for SOM peptide in this study. Subsequent experiments in the brain material obtained from these animals are now focusing on studying potential changes in hypothalamic estrogen and

months old) OVX female Wistar rats at different time points after E2 treatment. a

and ZT11 on both days (Figure 3B).

Tukey HSD). n=5 for each group.

progesterone receptor immunoreactivity.

**4. Discussion** 

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 GnRH priming mechanism may be particularly affected.

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