**Somatostatin in the Periventricular Nucleus of the Female Rat: Age Specific Effects of Estrogen and Onset of Reproductive Aging**

Eline M. Van der Beek, Harmke H. Van Vugt, Annelieke N. Schepens-Franke and Bert J.M. Van de Heijning *Human and Animal Physiology Group, Dept. Animal Sciences, Wageningen University & Research Centre The Netherlands* 

## **1. Introduction**

70 Sex Steroids

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Waxman AR, White RD, Williams TD, Lachey JL, Seeley RJ, Lowell BB and Elmquist JK (2005) Mice lacking ghrelin receptors resist the development of dietThe functioning of the growth hormone (GH) and reproductive axis is known to be closely related: both GH overexpression and GH-deficiency are associated with dramatic decreases in fertility (Bartke, 1999; Bartke et al, 1999; 2002; Naar et al, 1991). Also, aging results in significant changes in functionality of both axes within a similar time frame.

In the rat, GH secretion patterns are clearly sexually dimorphic (Clark et al, 1987; Eden et al, 1979; Gatford et al, 1998). This has been suggested to result mainly from differences in somatostatin (SOM) release patterns from the median eminence (ME) (Gillies, 1997; Muller et al, 1999; Tannenbaum et al, 1990). SOM is synthesized in the periventricular nucleus of the hypothalamus (PeVN) and controls in concert with GH-releasing hormone (GHRH) the GH release from the pituitary (Gillies, 1987; Tannenbaum et al, 1990; Terry and Martin, 1981; Zeitler et al, 1991). An altered GH status is reflected in changes in the hypothalamic SOM system. For instance, the number of SOM cells (Sasaki et al, 1997) and pre-pro SOM mRNA levels (Hurley and Phelps, 1992) in the PeVN were elevated in animals overexpressing GH.

Several observations suggest that SOM may also affect reproductive function directly at the level of the hypothalamus. SOM synthesis in the hypothalamus and its release from the ME fluctuate over the estrous cycle. (Estupina et al, 1996, Zorrila et al, , 1991). Central injections with SOM or a SOM analog (octreotide) decreased the number of gonadotropic cells in the pituitary (Lovren et al, 1998; Nestorovic et al 2002; 2003). Also, we previously showed that a single central injection with octreotide significantly attenuated the E2-induced Luteinizing Hormone (LH) surge and significantly decreased the activation of Gonadotropin Releasing Hormone (GnRH) cells in the hypothalamus of female rats (van Vugt et al, 2004).

Age-related changes in fertility and fecundity are associated with selective changes at the level of the ovary and uterus (Meredith and Butcher, 1985; Nass et al, 1984; te Velde et al, 1998; Wise, 1982), pituitary gland (Brito et al, 1994; DePaolo et al, 1986; Krieg et al, 1995; Nass et al, 1984; Wise, 1982), and hypothalamus (Rubin et al, 1994; Wise, 1982; Wise et al, 2002*).* Reproductive aging is characterized by changes in the length of the reproductive

Somatostatin in the Periventricular Nucleus of the Female Rat:

**2. Material & methods** 

the Wageningen University.

**2.2 Experimental design** 

sample was drawn.

(Van Vugt et al, 2008).

**2.3 Estrous cycle length** 

dark onset to confirm a proestrous lavage typing.

to 24%) and acyclicity (from 0% to 10%) increased.

**2.4 Cannulation and blood sampling** 

**2.1 Animals** 

Age Specific Effects of Estrogen and Onset of Reproductive Aging 73

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

To study changes in proestrous LH and P surge characteristics with age, 4-month-old ('young') and 8.5-month-old ('middle-aged') female rats with regular 4-day cycles were used for blood sampling and hormone analyses. Hourly blood samples were taken on proestrus to measure plasma LH and P profiles. To investigate pituitary LH and FSH responsiveness, a potent GnRH analog (Ovalyse; des-Gly10-GnRH-ethylamide, Upjohn, Ede, The Netherlands) was used on the following proestrus. Ovalyse (100 ng in 0.25 ml 0.9% NaCl (w/v) containing 1% BSA) was administered i.v. immediately after the first blood

Subsequently, a group of cycling females was ovariectomized (OVX, Van der Beek et al, 1999) at 4.5 (young) or 9 mo (middle-aged) of age, and given a single s.c. injection with estrogen at ZT3 on day 13 following OVX. Animals were perfused 2, 8, 26 or 32 hrs later, i.e. at ZT5 and ZT 11. Brains sections were stained for SOM peptide as described previously

Estrous cycles were monitored by daily vaginal lavage. Lavages were analyzed according to criteria described elsewhere (Freeman et al, 1994). In addition, receptive behavior (hopping and darting, ear wiggling and lordosis posture) was monitored daily. To this end, a naive male WU rat was introduced briefly in the female's home cage around ZT 11, 1 hr before

Cycle length was defined by the last two monitored cycles before sampling. Most females displayed regular 4-day estrous cycles (70%). Cycle lengthening was observed in 8.5 mo old females: regular 4-day cycles decreased from 70% to 45%, while both 5-day cycles (from 10%

The right jugular vein of female rats was cannulated to obtain stress-free blood samples (Steffens, 1969, Van der Beek et al 1999, Van Vugt et al, 2004). After a recovery period of at least five days, ten hourly blood samples of 170 l were taken on proestrus from ZT 5.5-14.5 for measurement of endogenous, preovulatory hormone profiles. To assess the amount of acutely releasable LH by the pituitary gland, ten hourly blood samples of 170 l were drawn from ZT 5.5-14.5 on the following proestrus, following an i.v. injection of the GnRH analog Ovalyse, just after the first sample. Blood samples were collected in heparinized, air-dried

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 to reproductive aging is unclear.

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 attenuation of the pituitary LH surge.

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

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

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 an attenuated LH surge in middle- aged rats.
