**4.3 Reproductive aging and the hypothalamus**

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

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

Somatostatin in the Periventricular Nucleus of the Female Rat:

involved in the regulation of both LH and GH release from the pituitary.

**4.4.1 Pathway 1: SOM projections to neurons in the OVLT/POA** 

**4.4.2 Pathway 2: SOM effects on LH release indirectly via NPY** 

and release, leading to the observed decreased LH surge (Figure 4-III).

**4.4.3 Pathway 3: SOM effects pituitary LH release indirectly** 

(pathway B in Figure 4-II).

Figure 4-IV).

Age Specific Effects of Estrogen and Onset of Reproductive Aging 81

therefore also be involved in the regulation of GH release from the pituitary (Herbison et al, 1994; Murray et al, 1999; Rage et al, 1993; Willoughby et al, 1987). Also, within the PeVN, a small number of SOM neurons co-express GABA (Tanaka et al, 1997). GHRH neurons in the ARC are inhibited by SOM neurons originating from either the PeVN or the ARC (McCarty et al, 1992; Lanneau et al, 2000; Tannenbaum et al, 1990; Willoughby et al, 1989). Neuropeptide-Y (NPY) terminals originating from the ARC project to the preoptic region and ME, in which some of the axons make synaptic contacts with GnRH cell bodies and processes (Smith and Jennes, 2003). Also, NPY cells may project to SOM cells within the PeVN. NPY may hence be

We showed that a centrally injected SOM analog decreased hypothalamic GnRH cell activation (Van Vugt et al, 2004), suggesting that SOM directly affects cells in the OVLT/POA. The fact that SSTRs were demonstrated in the OVLT/POA (Helboe et al, 1998; Schindler et al, 1996), and that lesions of the anterior hypothalamic area (including the PeVN) resulted in decreased SOM peptide levels in the POA (Epelbaum et al, 1977), suggests that SOM cells originating from the PeVN project to the OVLT/POA. Possibly, GnRH neurons themselves express SSTRs, so SOM may directly inhibit GnRH cell activation, leading to the supposed decrease in GnRH release, and hence to decreased LH release from the pituitary (pathway A in Figure 4-II). Alternatively, cells, other than GnRH-producing, in the OVLT/POA may contain SSTRs. Neurons in the periventricular POA that project to GnRH neurons at the time of the preovulatory LH surge (Le et al, 1997; 1999; 2001) are a likely candidates. Although not identified yet, GABA-ergic cells may be (one of) these neurons containing SSTRs and projecting to the GnRH neurons

NPY is very likely to influence the preovulatory LH surge: NPY synthesis and release are elevated just before the proestrous LH surge, and immunoneutralization of NPY prevents the steroid-induced LH surge. The effects of NPY on LH release may, at least in part, take place at the hypothalamic level, as NPY terminals synapse on GnRH cell bodies and processes (Smith and Jennes, 2003). As SSTRs were demonstrated on NPY cells in both the PeVN and ARC (Lanneau et al, 2000), SOM may inhibit NPY neurons activity, resulting in a decreased stimulating signal to GnRH cells, which in turn decreases GnRH cell activation

Besides the decreased LH surge, we also found decreased plasma GH concentrations following the centrally injected SOM analog (Van Vugt et al, 2004). SOM was shown to directly decrease LH release (Yu et al, 1997) and affect gonadotroph cell number and morphology (Lovren et al, 1998). Moreover, both gonadotrophs and somatotrophs express SSTRs. Hence, SOM may directly decrease both LH and GH release from the pituitary. The decrease in GH release leads to decreased IGF-I release, which may subsequently result in a decreased GnRH release from the ME (Miller et al, 2003; Zhen et al, 1997) (pathway C in

the LH surge appears to decrease with age (Matt et al, 1998). This is thought to be the result of a decreased activity of the GnRH system, a reduced responsiveness to GnRH signaling, and/or a reduction in cellular LH with age (Matt et al, 1998; Rubin et al, 2000). Also, the GnRH neuroterminal-glial-capillary unit in the ME may be affected, influencing the regulation of GnRH release (Yin et al, 2009). There are no indications that the number of pituitary GnRH receptors is affected with age in female mice (Belisle et al, 1990) and our results after Ovalyse® administration suggest that the responsiveness to (robust) GnRH signaling is still intact. Indeed, several studies demonstrated that on proestrus the number of activated GnRH neurons (Wise et al, 2002; Rubin et al, 1994) and endogenous GnRH release (Rubin et al, 2000) are reduced in middle-aged female rats. The activity of GnRH neurons is regulated by many different neural signals (Smith and Jennes, 2001) and several of the systems involved in the regulation of the GnRH surge are also affected with age (Wise et al, 2002; Sahu et al, 1998; Gore et al, 2002; Mills et al, 2002). Taken together, this suggests that the input onto GnRH neurons may change with age, resulting in less activated GnRH neurons and reduced GnRH release that together with a reduction in endogenous GnRH priming could indeed lead to an attenuated LH surge. Previous studies have proposed a contribution of the suprachiasmatic nucleus (SCN) in the attenuation of the LH release surge (Wise et al, 2002; Downs and Wise 2009). Yet, we did not find any significant changes in timing of the LH surge (i.e. LH surge onset and/or peak levels) in middle-aged rats. Since a clear delay in timing of the LH surge at the age of 7-10 months has only been shown in Sprague-Dawley rats (Sahu et al, 1998; Wise, 1982), the age at which changes in SCN output influence the LH surge mechanism may be strain specific.

#### **4.4 Effect of SOM on the reproductive axis**

Previous studies provide evidence for a proposed central role of hypothalamic SOM neurons in the functional interaction between the somatotropic and gonadotropic axis. Octreotide, given during the "critical period" of the day (i.e. just prior to surge onset), completely abolished the E2-induced LH surge and decreased GnRH cell activation (Van Vugt et al, 2004). Based on this, and the fact that SOM release may increase on proestrous afternoon (Estupina et al, 1983; Knuth et al, 1983; Zorilla et al, 1991), we hypothesize that in the cycling female rat, SOM release probably increases only after the "critical period", i.e. during the LH surge. Thus, we suggest that elevated levels of SOM on proestrous afternoon may be involved in the descending, rather than the ascending, phase of the preovulatory LH surge.

Our previous studies strongly suggest that SOM decreases LH release at least in part by decreasing hypothalamic GnRH neuron activation (Van Vugt et al 1994). However, the mechanism behind this action remains speculative. Moreover, indirect effects of SOM cannot be excluded, as SOM was demonstrated to directly affect gonadotropic cell number and morphology (Lovren et al. 1998). Here we propose three possible pathways via which SOM, originating from the PeVN, may affect GnRH neurons, resulting in a decreased LH release (see Figure 4).

The interactions between neurons in the hypothalamic areas involved in the regulation of LH (Preoptic Region, OVLT/POA) and GH (PeVN and Arcuate nucleus ARC) release are schematically depicted in Figure 4-I. GnRH neurons in the OVLT/POA are innervated by gamma-aminobutyric acid (GABA)-ergic cells, which are thought to be involved in the negative feedback of E2 on the LH surge (Miller et al, 2003; Zhen et al, 1997). These GABAergic cells originating from the OVLT/POA innervate SOM neurons in the PeVN and may

the LH surge appears to decrease with age (Matt et al, 1998). This is thought to be the result of a decreased activity of the GnRH system, a reduced responsiveness to GnRH signaling, and/or a reduction in cellular LH with age (Matt et al, 1998; Rubin et al, 2000). Also, the GnRH neuroterminal-glial-capillary unit in the ME may be affected, influencing the regulation of GnRH release (Yin et al, 2009). There are no indications that the number of pituitary GnRH receptors is affected with age in female mice (Belisle et al, 1990) and our results after Ovalyse® administration suggest that the responsiveness to (robust) GnRH signaling is still intact. Indeed, several studies demonstrated that on proestrus the number of activated GnRH neurons (Wise et al, 2002; Rubin et al, 1994) and endogenous GnRH release (Rubin et al, 2000) are reduced in middle-aged female rats. The activity of GnRH neurons is regulated by many different neural signals (Smith and Jennes, 2001) and several of the systems involved in the regulation of the GnRH surge are also affected with age (Wise et al, 2002; Sahu et al, 1998; Gore et al, 2002; Mills et al, 2002). Taken together, this suggests that the input onto GnRH neurons may change with age, resulting in less activated GnRH neurons and reduced GnRH release that together with a reduction in endogenous GnRH priming could indeed lead to an attenuated LH surge. Previous studies have proposed a contribution of the suprachiasmatic nucleus (SCN) in the attenuation of the LH release surge (Wise et al, 2002; Downs and Wise 2009). Yet, we did not find any significant changes in timing of the LH surge (i.e. LH surge onset and/or peak levels) in middle-aged rats. Since a clear delay in timing of the LH surge at the age of 7-10 months has only been shown in Sprague-Dawley rats (Sahu et al, 1998; Wise, 1982), the age at which changes in SCN output

Previous studies provide evidence for a proposed central role of hypothalamic SOM neurons in the functional interaction between the somatotropic and gonadotropic axis. Octreotide, given during the "critical period" of the day (i.e. just prior to surge onset), completely abolished the E2-induced LH surge and decreased GnRH cell activation (Van Vugt et al, 2004). Based on this, and the fact that SOM release may increase on proestrous afternoon (Estupina et al, 1983; Knuth et al, 1983; Zorilla et al, 1991), we hypothesize that in the cycling female rat, SOM release probably increases only after the "critical period", i.e. during the LH surge. Thus, we suggest that elevated levels of SOM on proestrous afternoon may be involved in the

Our previous studies strongly suggest that SOM decreases LH release at least in part by decreasing hypothalamic GnRH neuron activation (Van Vugt et al 1994). However, the mechanism behind this action remains speculative. Moreover, indirect effects of SOM cannot be excluded, as SOM was demonstrated to directly affect gonadotropic cell number and morphology (Lovren et al. 1998). Here we propose three possible pathways via which SOM, originating from the PeVN, may affect GnRH neurons, resulting in a decreased LH

The interactions between neurons in the hypothalamic areas involved in the regulation of LH (Preoptic Region, OVLT/POA) and GH (PeVN and Arcuate nucleus ARC) release are schematically depicted in Figure 4-I. GnRH neurons in the OVLT/POA are innervated by gamma-aminobutyric acid (GABA)-ergic cells, which are thought to be involved in the negative feedback of E2 on the LH surge (Miller et al, 2003; Zhen et al, 1997). These GABAergic cells originating from the OVLT/POA innervate SOM neurons in the PeVN and may

descending, rather than the ascending, phase of the preovulatory LH surge.

influence the LH surge mechanism may be strain specific.

**4.4 Effect of SOM on the reproductive axis** 

release (see Figure 4).

therefore also be involved in the regulation of GH release from the pituitary (Herbison et al, 1994; Murray et al, 1999; Rage et al, 1993; Willoughby et al, 1987). Also, within the PeVN, a small number of SOM neurons co-express GABA (Tanaka et al, 1997). GHRH neurons in the ARC are inhibited by SOM neurons originating from either the PeVN or the ARC (McCarty et al, 1992; Lanneau et al, 2000; Tannenbaum et al, 1990; Willoughby et al, 1989). Neuropeptide-Y (NPY) terminals originating from the ARC project to the preoptic region and ME, in which some of the axons make synaptic contacts with GnRH cell bodies and processes (Smith and Jennes, 2003). Also, NPY cells may project to SOM cells within the PeVN. NPY may hence be involved in the regulation of both LH and GH release from the pituitary.
