**2.1 Animals**

72 Sex Steroids

cycle. In female rats, for instance, the normal 4 or 5 day estrous cycle will lengthen with age and become irregular. This is followed by a period of repetitive pseudopregnancies and/or persistent estrus, while cyclicity ends with a state of persistent diestrus (Vom Saal, 1994). Although the general sequence of events during aging is predictable, the age at which the decline in fertility becomes evident varies considerably between individuals and rat strains (Vom Saal et al, 1994; Te Velde et al, 1998). Hence, especially during the initial stage when cyclicity is still regular, the relative contribution of the ovaries, pituitary and hypothalamus

One of the first, common changes appears to be an attenuation of the proestrous, ovulationinducing luteinizing hormone (LH) surge (Wise et al, 2002), which can be demonstrated even before estrous cycles become irregular (DePaolo et al, 1986). The latter is strongly associated with the age at which rats become acyclic (Nass et al, 1984). Previous research suggested that changes in ovarian hormone release (DePaolo et al, 1986; Lu et al, 1985), pituitary hormone storage and/or responsiveness to ovarian or hypothalamic signaling (Brito et al, 1994; Keizer et al, 2001; Matt et al, 1998), or changes in hypothalamic signaling (Rubin et al, 2000; Downs and Wise, 2009; Wise et al, 2002) may underlie the age-related

Evidence suggests that exposure to chronically elevated levels of circulating E2 during life advances the decline in fertility with age (Lu et al, 1981; Rodrigues et al, 1993). Moreover, it is known that E2 affects hypothalamic SOM content and release, although the literature is somewhat controversial on the precise role of E2 on SOM cell function (Baldino et al, 1988; Estupina et al, 1996; Knuth et al, 1983; Murray et al, 1999; Werner et al, 1988; Zorilla et al, 1991). Recent studies demonstrated a clear sex difference in the number and distribution of SOM peptide containing cells in the PEVN. In the female numbers were affected by

During the early phase of reproductive aging, normal (or even elevated) levels of plasma estradiol (E2), are correlated with a decline in somatotropic axis activity (Chandrashekar and Bartke, 1993; Vom Saal et al, 1994; Wilshire et al, 1995). In 14 months old rats, hypothalamic SOM peptide content as well as basal and KCl-stimulated SOM release from the hypothalamus were increased compared to young animals (Ge et al, 1989). Compared to young female rats, SOM peptide levels in the ME are decreased at 25-29 months of age (Takahashi et al, 1987), suggesting increased SOM release from the ME with age. Altogether, these data point to the hypothalamic SOM system as a potential candidate to mediate some

In the light of the data described above, we set out to study the effects of E2 exposure on hypothalamic SOM peptide levels at middle age when an attenuation of the LH surge can be found in regularly cycling females. To this end, we measured LH and P release in regularly 4-day cycling females at young (4 months) and middle-age (8.5 months) on proestrus as well as after a stimulus with a potent GnRH analog the following proestrus day. Subsequently, animals were ovariectomized to examine the effect of a physiological dose of E2 on SOMpeptide containing cells in the PeVN at selected time points following estrogen exposure. Using this approach, we aimed to gain more insight in the mechanisms underlying the interaction between the somatotropic and gonadotropic axis, i.e. a possible role for the hypothalamic SOM system. We hypothesize that SOM plays a role in the normal, physiological regulation of LH release in the female rat and suggest that changes in the response of PeVN SOM-ir with age may contribute to the hypothalamic changes that lead to

of the concurrent changes in the activity of the reproductive and GH axis with age.

ovariectomy and gonadal steroid treatment (Van Vugt et al, 2008).

to reproductive aging is unclear.

attenuation of the pituitary LH surge.

an attenuated LH surge in middle- aged rats.

Virgin female (n=60) and male (n=8) Wistar rats (HsdCpb:WU, Wistar Unilever) were obtained from Harlan (Horst, NL) at 9 to 10 weeks of age. Rats were group housed (4/cage) under regular light-dark cycles (L/D 12:12, lights on at 3:00 h defined as 'zeitgebertime' 0, ZT0) with free access to standard food pellets (Hope Farms B.V., Woerden, NL) and water. Animals were housed individually from 1 week before cannulation onwards. Young and Middle-aged females were obtained from the same batch to reduce variation between animals. All experiments were approved by the animal experimental committee (DEC) of the Wageningen University.
