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

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 (pathway B in Figure 4-II).

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

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

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

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 Figure 4-IV).

Somatostatin in the Periventricular Nucleus of the Female Rat:

in a decrease in plasma LH levels (see figure 2).

Age Specific Effects of Estrogen and Onset of Reproductive Aging 83

Although direct effects of SOM at the pituitary level in the regulation of the LH surge (pathway 3) cannot be excluded, in the cycling female rat this pathway seems very unlikely to be the primary one with respect to hypothalamic regulation of the preovulatory LH surge. We suggest that the direct effects of SOM at the level of the pituitary may be additional to the effects at the level of the hypothalamus concerning the proposed interaction with the reproductive axis. Also, the suggested role for NPY in the hypothalamic regulation of the LH surge in the female rat (pathway 2) is probably one of the factors in a complex regulatory mechanism. In the light of our own data and data from literature, we propose that the role of SOM in the regulation of the descending phase of the LH surge, may involve, at least, a combination of pathways 1 and 2. In the cycling female rat, elevated plasma concentrations of E2 and P on the day of proestrus may increase NPY levels in the ARC that, together with the removed inhibitory GABA-ergic tone (Smith and Jennes, 2003), stimulate GnRH cell activation, leading to the GnRH surge and, subsequently, the preovulatory LH surge. Secondly, the increased levels of gonadal steroids (Estupina et al, 1996, Van Vugt et al, 2008), and in addition, elevated levels of NPY (Rettori et al, 1990) may increase SOM release from the ME. Elevated concentrations of SOM, in turn, inhibit either neuron activity in the OVLT/POA, or NPY and its stimulating effects on GnRH neurons, or both, leading to decreased GnRH cell activation and subsequently release, finally resulting

Fig. 5. Proposed mechanism via which hypothalamic SOM may be involved in the regulation of the descending phase of the preovulatory LH surge in the female rat

In young rats, the rostrocaudal distribution profiles of SOM-ir cells within the PeVN were comparable between ZT5 as well as ZT11 on the two subsequent days after E2 treatment. In addition, the total SOM-ir fiber area, was consistently higher at ZT5 compared to ZT11 in these animals. These findings suggest that SOM peptide synthesized in the PeVN and released in the ME may be influenced by diurnal rhythms. Our data are supported by a study that reported a diurnal rhythm in SOM peptide content in the ME (Esquifino et al, 2004). Moreover, SOM peptide levels in the cortex, anterior hypothalamus and suprachiasmatic nucleus (SCN) (Fukuhara et al, 1993), and SOM release from the

**4.5 Age dependent effects of estrogen on PeVN SOM peptide** 

Fig. 4. Schematic drawings of the three proposed pathways via which SOM originating from the PeVN may decrease the LH surge. I: interactions between neurons in the OVLT/POA, PeVN and ARC as described in literature. II: direct effect of SOM on neurons in the OVLT/POA; A: directly on GnRH neurons; B: indirectly via cells projecting to GnRH neurons. III: indirect effect of SOM on GnRH neurons via NPY cells in the ARC. IV: indirect effect of SOM on GnRH neurons via the pituitary; C: direct effect of SOM on LH and GH cells; D: indirect effect of SOM on pituitary cells via GHRH neurons in the ARC. For more details: see text. Black circles represent SSTRs.

Alternatively, elevated SOM levels may inhibit GHRH neurons in the ARC (McCarty et al, 1992; Lanneau et al, 2000; Tannebaum et al, 1990; Willoughby et al, 1989), resulting in decreased GH release from the pituitary. As somatotroph and gonadotroph cell coexpression in the pituitary is maximal on the day of proestrus (Childs, 2000; Childs et al, 2000; 1994), a decreased activation of GH cells may lead to decreased activity of LH cells, consequently resulting in a decreased LH release. In addition, decreased IGF-I levels, due to decreased plasma GH concentrations, may lead to both decreased LH release from the pituitary (Kanematsu et al, 1991) and decreased GnRH release from the ME (Miller et al 2003; Zhen et al, 1997) (pathway D in Figure 4-IV).

Fig. 4. Schematic drawings of the three proposed pathways via which SOM originating from the PeVN may decrease the LH surge. I: interactions between neurons in the OVLT/POA, PeVN and ARC as described in literature. II: direct effect of SOM on neurons in the OVLT/POA; A: directly on GnRH neurons; B: indirectly via cells projecting to GnRH neurons. III: indirect effect of SOM on GnRH neurons via NPY cells in the ARC. IV: indirect effect of SOM on GnRH neurons via the pituitary; C: direct effect of SOM on LH and GH cells; D: indirect effect of SOM on pituitary cells via GHRH neurons in the ARC. For more

Alternatively, elevated SOM levels may inhibit GHRH neurons in the ARC (McCarty et al, 1992; Lanneau et al, 2000; Tannebaum et al, 1990; Willoughby et al, 1989), resulting in decreased GH release from the pituitary. As somatotroph and gonadotroph cell coexpression in the pituitary is maximal on the day of proestrus (Childs, 2000; Childs et al, 2000; 1994), a decreased activation of GH cells may lead to decreased activity of LH cells, consequently resulting in a decreased LH release. In addition, decreased IGF-I levels, due to decreased plasma GH concentrations, may lead to both decreased LH release from the pituitary (Kanematsu et al, 1991) and decreased GnRH release from the ME (Miller et al

details: see text. Black circles represent SSTRs.

2003; Zhen et al, 1997) (pathway D in Figure 4-IV).

Although direct effects of SOM at the pituitary level in the regulation of the LH surge (pathway 3) cannot be excluded, in the cycling female rat this pathway seems very unlikely to be the primary one with respect to hypothalamic regulation of the preovulatory LH surge. We suggest that the direct effects of SOM at the level of the pituitary may be additional to the effects at the level of the hypothalamus concerning the proposed interaction with the reproductive axis. Also, the suggested role for NPY in the hypothalamic regulation of the LH surge in the female rat (pathway 2) is probably one of the factors in a complex regulatory mechanism. In the light of our own data and data from literature, we propose that the role of SOM in the regulation of the descending phase of the LH surge, may involve, at least, a combination of pathways 1 and 2. In the cycling female rat, elevated plasma concentrations of E2 and P on the day of proestrus may increase NPY levels in the ARC that, together with the removed inhibitory GABA-ergic tone (Smith and Jennes, 2003), stimulate GnRH cell activation, leading to the GnRH surge and, subsequently, the preovulatory LH surge. Secondly, the increased levels of gonadal steroids (Estupina et al, 1996, Van Vugt et al, 2008), and in addition, elevated levels of NPY (Rettori et al, 1990) may increase SOM release from the ME. Elevated concentrations of SOM, in turn, inhibit either neuron activity in the OVLT/POA, or NPY and its stimulating effects on GnRH neurons, or both, leading to decreased GnRH cell activation and subsequently release, finally resulting in a decrease in plasma LH levels (see figure 2).

Fig. 5. Proposed mechanism via which hypothalamic SOM may be involved in the regulation of the descending phase of the preovulatory LH surge in the female rat

#### **4.5 Age dependent effects of estrogen on PeVN SOM peptide**

In young rats, the rostrocaudal distribution profiles of SOM-ir cells within the PeVN were comparable between ZT5 as well as ZT11 on the two subsequent days after E2 treatment. In addition, the total SOM-ir fiber area, was consistently higher at ZT5 compared to ZT11 in these animals. These findings suggest that SOM peptide synthesized in the PeVN and released in the ME may be influenced by diurnal rhythms. Our data are supported by a study that reported a diurnal rhythm in SOM peptide content in the ME (Esquifino et al, 2004). Moreover, SOM peptide levels in the cortex, anterior hypothalamus and suprachiasmatic nucleus (SCN) (Fukuhara et al, 1993), and SOM release from the

Somatostatin in the Periventricular Nucleus of the Female Rat:

**7. References** 

1999; 222: 113-123.

132: 1482-1488.

Age Specific Effects of Estrogen and Onset of Reproductive Aging 85

Baldino F, Jr., Fitzpatrick-McElligott S, O'Kane TM, Gozes I. Hormonal regulation of

Bartke A. Role of growth hormone and prolactin in the control of reproduction: what are we learning from transgenic and knock-out animals? Steroids 1999; 64: 598-604. Bartke A, Chandrashekar V, Bailey B, Zaczek D, Turyn D. Consequences of growth hormone (GH) overexpression and GH resistance. Neuropeptides 2002; 36: 201-208. Bartke A, Chandrashekar V, Turyn D, Steger RW, Debeljuk L, Winters TA, Mattison JA,

Beaudet A, Greenspun D, Raelson J, Tannenbaum GS. Patterns of expression of SSTR1 and

relationship to neuroendocrine function. Neuroscience 1995; 65: 551-561. Belisle S, Bellabarba D, Lehoux JG. Hypothalamic-pituitary axis during reproductive aging

Berelowitz M, Dudlak D, Frohman LA. Diurnal variation in release of somatostatin-like

Brann DW, Mahesh VB. The aging reproductive neuroendocrine axis. Steroids 2005; 70:273-283 Brito AN, Sayles TE, Krieg RJ, Matt DW. Relation of attenuated proestrous luteinizing

Chandrashekar V, Bartke A. Effects of age and endogenously secreted human growth

Chern BY, Chen YH, Hong LS, LaPolt PS. Ovarian steroidogenic responsiveness to

Childs GV, Unabia G, Wu P. Differential expression of growth hormone messenger

Childs GV. Growth hormone cells as co-gonadotropes: partners in the regulation of the

Childs GV, Unabia G, Rougeau D. Cells that express luteinizing hormone (LH) and follicle-

Clark RG, Carlsson LM, Robinson IC. Growth hormone secretory profiles in conscious

reproductive system. Trends.Endocrinol.Metab. 2000; 11: 168-175.

releasing hormone responsiveness. Eur J Endocrinol 1994; 130:540-544. Buijs RM, Pool CW, Van Heerikhuize JJ, Sluiter AA, Van der Sluis PJ, Ramkema M, Van der

Danilovich NA, Croson W, Wernsing DR, Kopchick JJ. Effects of growth hormone overexpression and growth hormone resistance on neuroendocrine and reproductive functions in transgenic and knock-out mice. Proc.Soc.Exp.Biol.Med.

SSTR2 somatostatin receptor subtypes in the hypothalamus of the adult rat:

immunoreactivity from incubated, rat hypothalamus and cerebral cortex.

hormone surges in middle-aged female rats to in vitro pituitary gonadotropin-

Woude TP, Van der Beek E. Antibodies to small transmitter molecules and peptides: production and application of antibodies to dopamine, seretonin, GABA, vasopressin, vasoactive intestinal peptide, neuropeptide Y, somatostatin and

hormone on the regulation of gonadotropin secretion in female and male transgenic mice expressing the human growth hormone gene. Endocrinology 1993;

exogenous gonadotropin stimulation in young and middle aged female rats. Proc

ribonucleic acid by somatotropes and gonadotropes in male and cycling female

stimulating hormone (FSH) beta-subunit messenger ribonucleic acids during the estrous cycle: the major contributors contain LH beta, FSH beta, and/or growth

somatostatin messenger RNA. Synapse 1988; 2: 317-325.

in mice. Mech Ageing Dev 1990; 52:207-217.

substance P. Biomedical Research 1989; 10: 213-221.

Endocrinology 1982; 110: 2195-2197.

Soc Exp Biol Med 2000; 224:285 291.

rats. Endocrinology 2000; 141: 1560-1570.

hormone. Endocrinology 1994; 134: 990-997.

female rats. J.Endocrinol. 1987; 114: 399-407.

hypothalamus (Berelowitz et al, 1982) were demonstrated to show circadian rhythmicity and suggest that the SCN may play a role in this diurnal change in SOM peptide transport from the PeVN to the ME. Thus, our data suggest that in the young female Wistar rat SOM cells in the PeVN are influenced by at least the SCN and E2. E2 may affect intrahypothalamic SOM projections within the PeVN or to other hypothalamic areas that contain SOM receptors (Beaudet et al, 1995; Hervieu et al, 1999), whereas SOM release from the ME may be influenced by the SCN. SOM content and release from hypothalamic explants is influenced by sex and age (Ge et al, 1989). The rostro-caudal distribution pattern of SOM-ir cells and the total number of SOM-ir cells in the PeVN was different in the middle-aged compared to young rats, but only 2 h after E2 treatment. These findings suggest that with age, E2 may become more crucial for the synthesis and/or storage of SOM peptide in the PeVN and affect the diurnal change in SOM levels within the PeVN.

The function of a diurnal change in SOM levels in the PeVN remains speculative. A few studies reported more pronounced GH secretory bursts in cycling female rats after the onset of darkness (Clark et al, 1987; Pincus et al, 1996), suggesting that the shift in the rostro-caudal SOM cell distribution at ZT11, i.e. just before dark onset, may reflect this shift in GH secretion pattern. Although to our knowledge no data exist on light/dark-related GH secretory patterns during aging, mean plasma GH levels and mean peak GH levels were found to be decreased already in 11 month old compared to young females (Takahashi et al, 1987). Taking these findings into consideration, we suggest that the changes in SOM-ir levels within the PeVN may translate into changes in GH release patterns during aging in female rats.
