View See Gore 2001, Ojeda Et Al

MIAMI UNIVERSITY The Graduate School

CERTIFICATE FOR APPROVING THE DISSERTATION

We hereby approve the Dissertation

Jill Marie Russell

Candidate for the Degree:

Doctor of Philosophy

Director Phyllis Callahan, PhD

Reader James Janik, PhD

Reader Paul James, PhD

Graduate School Representative Emily Murphree, PhD

ABSTRACT

THE EFFECT OF STEROIDS ON NEUROENDOCRINE FUNCTION IN IMMATURE RATS

By Jill Marie Russell

The hypothesis tested was that a developmental difference in the effects of estrogen and progesterone on nitric oxide and dopamine influences the steroid-induced luteinizing hormone and prolactin surges. I investigated the stimulatory effects of steroid hormones (estrogen and progesterone) on luteinizing hormone and prolactin secretion in prepubertal (4 weeks), peripubertal (6 weeks) and sexually mature (12 & 16 weeks) female rats. Additionally, the effects of L-arginine, on the steroid induced luteinizing hormone and prolactin secretions were examined in peripubertal female rats. I also investigated the regulatory role of steroid hormones on nitric oxide synthase and tyrosine hydroxylase levels in the hypothalamus of pre-pubertal (4 weeks) and sexually mature (12 weeks) female rats. For each age, intact, ovariectomized and ovariectomized plus steroid replaced females were examined. These studies advanced our understanding of the neural regulation of puberty and cyclicity by examining the regulatory role of ovarian steroids on nitric oxide and dopamine and its regulatory role on puberty and the development of cyclicity in females. Importantly, these studies investigated the changes that occur during pubertal development.

THE EFFECT OF STEROIDS ON NEUROENDOCRINE FUNCTION IN IMMATURE RATS

A DISSERTATION

Submitted to the Faculty of

Miami University in partial

fulfillment of the requirements

for the degree of

Doctor of Philosophy

Department of Zoology

Jill Marie Russell

Miami University

Oxford, OH

2004

Dissertation Director: Phyllis Callahan, PhD Table of Contents

Certificate for Approving the Dissertation Abstract Title page………………………………………………………………………………..i Table of Contents………………………………………………………………………..ii List of tables……………………………………………………………………………..iii List of figures……………………………………………………………………………iv Dedication………………………………………………………………………………..v Acknowledgements………………………………………………………………………vi Chapter 1…………………………………………………………………………………1 Chapter 2…………………………………………………………………………………15 Chapter 3…………………………………………………………………………………39 Chapter 4…………………………………………………………………………………70

ii List of Tables

Table 2.1..………………………………………………………………………………..28

iii List of Figures

Figure 1.1…………………………………………………………………………………10 Figure 2.1..………………………………………………………………………………..31 Figure 2.2…………………………………………………………………………………32 Figure 2.3…………………………………………………………………………………33 Figure 2.4…………………………………………………………………………………34 Figure 2.5…………………………………………………………………………………35 Figure 2.6…………………………………………………………………………………36 Figure 2.7…………………………………………………………………………………37 Figure 2.8…………………………………………………………………………………38 Figure 3.1.a……………………………………………………………………………….61 Figure 3.1.b……………………………………………………………………………….62 Figure 3.2.a……………………………………………………………………………….63 Figure 3.2.b……………………………………………………………………………….64 Figure 3.3…………………………………………………………………………………65 Figure 3.4.a……………………………………………………………………………….66 Figure 3.4.b……………………………………………………………………………….67 Figure 3.5.a……………………………………………………………………………….68 Figure 3.5.b……………………………………………………………………………….69

iv Dedication

This work is dedicated to my family. To Mom & Dad; who persevered and always saw the potential in me. To Vickie, Sam, Tim, Suzie & Gardiner; who took care of me. To Dave; who would not let me quit. To Leslie, Amanda & Paul; for accepting me and enriching my life. To Katlyn and Dylan; who gave my life purpose and motivated me to better myself. You are my inspiration. I could not have accomplished this work without your unending love and support. Thank you.

v Acknowledgments

To my co-advisors, Phyllis Callahan & Jim Janik, a big thank you. I would not have stayed in graduate school, if you had not taken me into your lab. The level of scientific research in your lab is superior. I learned valuable experimental techniques from you. You have been great mentors and friends. Thank you for keeping me on track and dealing with my disabilities. I know it has not been easy for you, and I appreciate your patience and tutelage. To Paul James, for teaching me how to run Westerns and for keeping me in your lab even after I broke your equipment. To Paul Harding, for your regular words of encouragement and sense of humor. To Emily Murphree, for surviving the brain storming sessions with Phyllis and me. Thanks for running all the stats all these years, and for having patience with us.

vi CHAPTER 1

General Introduction Puberty Puberty is a dynamic stage in the mammalian female life cycle, which is typically associated with increases in body size and the development of reproductive function. In the female rat, puberty is characterized by several events including vaginal opening, onset of receptive behavior patterns (soliciting and lordosis), initiation of cyclic pituitary gland hormone secretion, and ovulation (for review see Gore 2001, Ojeda et al. 1994). The hypothalamus is the site of vital neurochemical activity during both the onset of puberty and in the regulation of the adult pattern of cyclic pituitary secretory patterns which stimulate the release of eggs from the ovaries on a consistent basis (Clough et al. 1983, Grumbach 2002). These cyclical hormone patterns are undetected in the immature female rat until the onset of puberty (for review see Ojeda et al. 1983b, Ojeda et al. 1994). The anatomical and physiological changes that occur within the female mammal during puberty are dependent upon neural changes during embryonic and prepubertal development that lead to the activation of gonadotropin releasing hormone (GnRH) neurons in the hypothalamus (Gore 2001). The ovaries and pituitary gland of immature rats function normally when transplanted into mature animals, indicating that these organs are functionally mature, even prior to puberty (Debeljuk et al. 1972). GnRH stimulates the release of the reproductive hormones, follicle stimulating hormone (FSH) and luteinizing hormone (LH), from the anterior pituitary gland. FSH stimulates the initial development and maturation of ovarian follicles, as well as the secretion of estrogen (E2) by the follicles. In response to positive feedback effects of E2, an LH surge occurs and the increased LH induces the first ovulation, marking the onset of sexual maturity (Gore 2001, Grumbach 2002). In the rat, a concomitant prolactin (PRL) surge occurs, and serves a luteotropic function (Freeman et al. 2000). This cyclic pattern of hormonal secretion continues during the reproductive lifespan of the adult female. Postnatal prepubertal development of the female rat can be divided into four phases: (1) a neonatal period initiated at birth and ending on postnatal day 7; (2) an infantile period that extends from days 7 – 21; (3) a juvenile period that ends around days 30-32 and; (4) a

1 peripubertal period of variable duration that culminates with the occurrence of the first ovulation (around day 38 for most laboratory stocks) (for review see (Ojeda et al. 1994).

During the juvenile period of development there are sporadic LH spikes. The magnitude of these spikes becomes attenuated during the juvenile period due to E2 negative feedback, which keeps circulating gonadotropin levels low. In order to induce an LH surge during the early

juvenile period, E2 doses twice as high as those on proestrus must be administered (for review see Ojeda et al. 1994). As juvenile development continues, E2 levels stimulate norepinephrine (NE) and glutamate production in the hypothalamus, as well as increases in neuronal growth and synaptic connections, and increases in uterine growth and intrauterine fluid. Norepinephrine and dopamine (DA) turnover increase and pituitary DA receptor sensitivity decreases (Hohn et al. 1979, Lamberts et al. 1981, Raum et al. 1980). Blockade of catecholamine synthesis in late juvenile (30 day old) rats reduces LH levels, suggesting that catecholamines have a stimulatory effect on GnRH during puberty (Moguilevsky et al. 1995). While gonadotropin levels are low during juvenile development, PRL levels increase. PRL stimulates follicular growth, and increases steroid sensitivity to LH and FSH via interaction with LH receptors in the ovary (Advis et al. 1979). These levels of PRL also activate dopaminergic neurons, increasing DA release, which desensitizes pituitary DA receptors. PRL begins to be secreted in a variable, yet adult type pattern, and the ovary grows. This pattern of release is characterized by discharges occurring approximately every 3 hours. The most prominent PRL secretions occur at midafternoon and during the early morning hours.

As E2 levels continue to rise and after E2 and progesterone (P4) levels have remained high for an extended period, a GnRH surge is induced, via steroid positive feedback. Whether this is a direct effect, or one mediated by intermediate factors is still unclear. This shift from negative feedback to positive feedback sets in motion the events of puberty and the beginning of cyclical hormone secretion. The nocturnal increases in PRL levels disappear, but the afternoon surge of both PRL and LH become even more pronounced (Ojeda et al. 1983a, Ojeda et al. 1994). It is believed that the shift in LH pulses is due to the final maturation of the GnRH circuitry, because GnRH is the primary stimulator of LH secretion. Activation of the adult pattern of GnRH, LH and PRL secretion is believed to be in response to both rising levels of stimulatory inputs such as

E2, glutamate (via NMDA receptors) and nitric oxide (NO), as well as a decline in inhibitory inputs, i.e. hypothalamic GABA and DA (Freeman et al. 2000, Gore 2001, Gore et al. 2002).

2 However, the exact mechanisms controlling these pituitary hormone surges and the initiation of puberty are unknown. For reviews of the mechanisms involved in pubertal development see (Advis et al. 1979, Andrews et al. 1980, Ojeda et al. 1983a, Ojeda et al. 2003, Ojeda et al. 1994, Urbanski et al. 1987). Once diurnal patterns of LH release become established, a new cascade of events develops that lead to the first preovulatory surge and the first ovulation. This cascade has caused many experts to divide the process of puberty into different phases based on morphological criteria. Puberty in the female rat can be divided into the following phases:

Anestrous is the phase of puberty during which the changes in the mode of LH release begin to occur (around age 30 days, and a wet uterine weight of less than 100 mg). No

intrauterine fluid can be detected at this stage, and the vagina is always closed. Serum E2 levels increase markedly between anestrous and proestrus, reaching about 80 pg/ml during the morning of late proestrus (Advis et al. 1979, Andrews et al. 1980).

Proestrus occurs when animals have larger uteri with intraluminal fluid and closed vaginae. Uterine fluid becomes apparent for the first time during early proestrus (Advis et al. 1979). The uterus is “ballooned” with a wet weight greater than 200 mg. Most

ovaries have large follicles. At noon on the proestrus day in the rat, E2 levels peak and stimulate the release of GnRH and LH, due to positive feedback, leading to ovulation. Nitric oxide also acts on GnRH nerve terminals stimulating GnRH release from the median eminence into the capillary beds of the pituitary portal vessels. The sustained

increase in E2 concentration exerts a negative feedback effect on the pituitary and hypothalamus so GnRH and LH levels begin to decline. Estrus is the first day of ovulation, when uterine fluid is no longer present, and fresh corpora lutea can be seen. The vagina is open and cornified cells are apparent in the vaginal epithelium.

Diestrus is the phase when leukocytes are predominant in the vagina and the corpora lutea are mature within the ovaries.

Following the first ovulation, the female begins an estrous cycle which stimulates ovulation every 4-5 days (Butcher et al. 1974). Neurochemical, neuroendocrine and systemic

3 factors are involved in coordinating the estrous cycle throughout the reproductive lifetime. I examined the effect of ovarian steroid hormones on LH and PRL neuroregulatory mechanisms in prepubertal and peripubertal female rats, focusing on the sensitivity of hypothalamic NO and DA to E2 and P4. A summary of the factors that are involved in the regulation of LH and PRL are depicted in Figure 1.1.

Luteinizing Hormone (LH) The primary stimulator of LH secretion is hypothalamic GnRH. GnRH is released from the hypothalamus in a pulsatile manner, and longer pulse intervals lead to a decrease in LH synthesis and release. Prior to ovulation, GnRH pulses increase and stimulate the release of LH (Etgen et al. 1999). The interaction between ovarian steroids and hypothalamic neurotransmitters/neuromodulators is believed to be responsible for the synchronized activation of LH secretion. Once released, GnRH travels through the hypothalamic-pituitary portal system and acts directly on GnRH receptors on the gonadotrophs in the anterior pituitary gland. When GnRH binds to receptors on the surface of gonadotrophs, LH is released into the blood and travels to the ovaries, inducing ovulation. Both the rate of LH synthesis and secretion are under the control of GnRH. While it is clear that estrogen regulates GnRH neuronal activity through indirect mechanisms, it may also act directly on GnRH neurons, probably through the β estrogen receptor subtype (ER-β) (for review see Herbison and Pape, 2001).

Estrogen and Progesterone (E2 and P4) Estrogen, the dominant female hormone, promotes development and maintenance of female reproductive structures and secondary sex characteristics, influences body fluid and electrolyte balance as well as increases protein anabolism. Progesterone acts synergistically with

E2 to prepare the endometrium for implantation of the fertilized ovum and mammary glands for milk secretion. There is a 2-3 fold increase in serum P4 observed before proestrus that may have a role in facilitating the stimulatory effect of E2 on GnRH release (Advis et al. 1979, Andrews et al. 1980). Administration of E2 to pubertal female rats results in the advancement of ovulation (Ramirez et al. 1965), and an increase in serum LH levels (Caligaris et al. 1972 , Clough and Rodriguez-Sierra, 1983) in intact and ovariectomized rats. In adult female rats, gonadal steroid hormone administration induced both LH and PRL surges from 1 day to 50 weeks following

4 ovariectomy (Heimke et al. 1987). Physiological amounts of E2 and P4 administered subcutaneously in a pattern similar to that naturally occurring on days 1 and 3 of the estrous cycle can induce LH and PRL surges, and has been used to mimic the normal estrous cycle (Clough et al. 1983).

Prolactin (PRL) PRL is essential for mammary growth and development, milk synthesis and initiating and maintaining lactation in mammals (Bole-Feysot et al. 1998, Neill et al. 1994). It also serves as a luteotropic factor in the rat by regulating progesterone production in cells of the corpus luteum (Bole-Feysot et al. 1998). Important to my research, PRL also accelerates the onset of puberty in females by acting on both the central nervous system and ovary (Ojeda 1983), where it enhances the ovarian responsiveness to gonadotropins (Advis et al. 1979).

Dopamine, synthesized in the arcuate nucleus of the hypothalamus, is the primary regulator of PRL release (Ben-Jonathan et al. 2001), providing tonic, inhibitory control over PRL secretion via tuberoinfundibular dopaminergic (TIDA) neurons. This inhibitory effect of DA is

disrupted by E2 on the day of the PRL surge (for review see (Freeman et al. 2000). TIDA neurons send short axonal projections to the median eminence of the hypothalamus and release dopamine into the portal blood. Dopamine travels to the anterior pituitary lactotrophs, where it

binds to D2 receptors and tonically inhibits PRL release (for review see Ben-Jonathan and Hnasko, 2001). PRL secretion is further regulated by negative feedback effects of PRL on the TIDA neurons; increased PRL stimulates TIDA neuronal activity by binding with PRL receptors on these dopaminergic neurons (for review see Ben-Jonathan and Hnasko, 2001). Although increased levels of GnRH correlate with the release of PRL, there are no GnRH receptors on the lactotrophs. However, it has been postulated that GnRH stimulates the co-release, with LH, of Angiotensin II from gonadotrophs and it is the paracrine action of Angiotensin II that mediates the concomitant release of PRL (Jones et al. 1988, Kubata et al. 1990). Nitric oxide has also been shown to influence PRL release but it has yet to be established if NO mediation of PRL release is primarily at the level of the hypothalamus or the lactotroph itself.

In the absence of E2, plasma PRL levels are low and relatively stable throughout the day

(Caligaris et al. 1974). E2 initiates small daily diurnal PRL surges (Caligaris 1977). In addition to its direct effects on the pituitary to increase PRL gene transcription, PRL synthesis and

5 secretion and the responsiveness of the lactotroph to stimulating and inhibiting factors, E2 also has a profound influence on DA neurons particularly TIDA neurons in the hypothalamus (for review see (Hou et al. 2003). Estrogen increases the concentration of tyrosine hydroxylase (TH), the rate limiting enzyme in catecholamine biosynthesis, and induces c-fos expression in TH- immunoreactive cells, indicating neural activation. Estrogen also reduces the number of D2 receptors and induces down-regulation of G proteins involved in the response to DA. Tonically, high levels of E2 also inhibit PRL release. It is known that the rising levels of ovarian steroids, which initiate the LH and PRL surges, affect both DA (see Freeman et al. 2000 for review) and GnRH, but the exact mechanism of action has yet to be determined. One important mediator of

E2 activity is NO, which is known to affect DA, GnRH, LH and PRL release (Grumbach 2002, Kriegsfeld et al. 2002, Velardez et al. 2003).

Nitric Oxide (NO) Nitric oxide (NO) has an important role in the control of reproduction. NO is formed from the substrate L-arginine (L-arg) via the action of the enzyme nitric oxide synthase (NOS) (Bredt 1999). Neuronal NOS (nNOS) is a constitutive, calcium and calmodulin dependent enzyme that can produce small amounts of NO rapidly and transiently in response to various stimulants. Once formed, NO diffuses to nearby neurons where it mediates its effects by influencing the function of second messengers, such as cGMP, and thus modulates cellular responses (Dawson et al. 1994, Krumenacker et al. 2004). There are three isoforms of NOS, but nNOS is the major form expressed in the central nervous system, including the hypothalamic regions known to regulate pituitary function (Bhat et al. 1995). That nNOS is the major NOS isoform responsible for NO production in the hypothalamus is further supported by the findings that NOS activity in the hypothalamus of endothelial NOS knock-out mice is normal, but eliminated in the hypothalamus of nNOS knockout mice (Hara et al. 1996).

E2 is an important regulator of NOS in the hypothalamus. Estrogen treatment up-regulates NADPH (NO cofactor)-diaphorase staining and nNOS mRNA in the rat hypothalamus (Ceccatelli et al. 1996, Herbison et al. 1996, Okamura et al. 1995, Okamura et al. 1994). Brann, et al. (Brann et al. 1997) reported that n-NOS mRNA and protein levels in the hypothalamus of the rat were increased on the afternoon of proestrus, providing evidence for a cyclical regulation of NO by steroids. Sahu (Sahu 1998) reported that NOS mRNA levels declined with age in

6 middle-aged (8-10 months old) female rats, indicating an age-related and possibly steroid dependant, change in NOS activity. In humans, E2 also induces enzymatic activity of

constitutive NOS and E2 replacement therapy in post-menopausal women significantly increases circulating nitrite and nitrate levels (Lopez-Jaramillo et al. 1999). Results from these studies support the hypothesis that NOS sensitivity to steroid hormones, i.e. both protein and activity levels, changes with developmental stage and exposure. GnRH neurons in the hypothalamus are surrounded by NOS-immunopositive neurons, suggesting that NO may regulate GnRH neurons (Bhat et al. 1996, Bredt 1999, Grossman et al. 1994, Herbison et al. 1996). Furthermore, this evidence suggests that NO plays a major role in the induction of both the pre-ovulatory LH and PRL surges in cycling female rats as well as the increase induced by steroids in ovariectomized (OVX) rats (Aguan 1996, Bonavera et al. 1994, Bonavera et al. 1993). This is important to the current study because GnRH mediates pituitary hormone release.

In mature, ovariectomized, E2-primed rats given L-Arginine, both the magnitude and duration of the LH surge were amplified when compared with ovariectomized rats receiving steroids alone (Bonavera et al. 1996). Peak LH levels in these rats were 17- to 20-fold higher than the basal levels in the morning, which is similar to the pre-ovulatory LH surge seen on the afternoon of proestrus in intact females (Bonavera et al. 1993, Kalra et al. 1971). Bonavera, et al. (1993, 1996) hypothesized that L-Arginine supplementation alleviated a deficiency of NO in mature, ovariectomized, steroid-treated rats. It is important to note that NO production in the hypothalamus was unchanged in ovariectomized rats, but when they received steroid replacement, there was a rise in hypothalamic NO (Bonavera et al. 1996). Increased substrate availability during the pre-surge period would increase NO production. With increased NO, it is likely there is increased sensitivity to stimuli that trigger the cascade of neurochemical events that produce the PRL and LH surges. Nitric oxide also appears to be involved in mediating the pre-ovulatory PRL surge by regulating dopaminergic inhibition of PRL secretion. NO prevents the afternoon fall of TIDA activity, which is essential for the E2 induced afternoon PRL surge (Yen et al. 1999). However, it is not clear whether the effect of NO is solely exerted at the level of the anterior pituitary gland to directly affect PRL secretion, or at the hypothalamus to regulate the secretion of PRL- releasing and/or inhibitory factors. Brunetti, et al. (1995) reported that NO donors enhanced

7 basal and interleukin-1ß-induced PRL release in vitro, while NOS inhibitors decreased this induced PRL release. Other in vivo studies support a stimulatory role for NO in the control of PRL secretion, e.g., subcutaneous injection of a NOS inhibitor markedly attenuated the pre- ovulatory PRL surge on the afternoon of proestrus in the adult rat (Bonavera et al. 1994). Similarly, systemic administration of L-Arginine stimulates PRL secretion in humans (Rakoff et al. 1973).

SIGNIFICANCE The purpose of this study was to examine the sensitivity to gonadal steroid hormones of the hypothalamic neuroregulatory mechanisms that control PRL and LH secretion. A major focus was on the role of nitric oxide in mediating the steroid induced pre-ovulatory LH and PRL surges in sexually immature female rats. Hormone surges were examined in intact and ovariectomized animals following steroid replacement. In Chapter 1, the NO levels were increased pharmacologically by L-arginine supplementation in a group of ovariectomized rats receiving gonadal steroid replacement. The effects of the steroids, in the absence and presence of NO supplementation, were determined. These experiments were performed on peripubertal (6 week old), ovariectomized females because the neural changes that occur during pubertal development are nearly completed by this age (Gore 2002). Sexually mature, ovariectomized females were used as comparison controls. In Chapter 2, the neuroregulatory mechanisms controlling PRL secretion during pubertal development were further examined. The PRL response to gonadal steroid hormones was determined in ovariectomized, prepubertal (4 week old) females. The sensitivity of hypothalamic NOS to steroid hormones was also examined by quantifying NOS levels. Finally, hypothalamic TH levels were determined as an indicator of the sensitivity of hypothalamic dopaminergic neurons to steroid hormone regulation. Prepubertal, intact and ovariectomized females, as well as adult, ovariectomized females that served as comparison controls, were examined. These studies are among the first to specifically address the regulation of PRL secretion in pubertal development and advance our understanding of the neural regulation of puberty and cyclicity.

8 Figure 1.1

This figure is a summary of some of the factors that regulate the secretion of LH and PRL. This

research focused on the role of the ovarian steroids, estrogen (E2) and progesterone on nitric oxide (NO), which affects the secretion of both LH and PRL. The role of dopamine (DA), the primary, inhibitory regulator of PRL secretion, in the development of PRL secretion during puberty, was also examined.

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Raum W, Glass A and Swerdloff R (1980) Changes in hypothalamic catecholamine neurotransmitters and pituitary gonadotropins in the immature female rat: Relationships to the gonadostat theory of puberty onset. Endocrinology 106, 1253-1258.

Sahu A (1998) Absence of increased hypothalamic nitric oxide synthase gene expression during the preovulatory LH surge in middle-aged rats. NeuroReport 9, 4019=4023.

Urbanski H and Ojeda S (1987) Neuroendocrine mechanisms controlling the onset of female puberty. Reproductive Toxicology 1, 129-138.

Velardez M, del Carmen Diaz M, Lasaga M, Franchi A and Duvilanski B (2003) Estrogen decreases the sensitivity of anterior pituitary to the inhibitory effect of nitric oxide on prolactin release. Hormone Research 60, 111-115.

Yen S and Pan J (1999) Nitric oxide plays an important role in the diurnal change of tuberoinfundibular dopaminergic neuronal activity and prolactin secretion on ovariectomized, estrogen/progesterone-treated rats. Endocrinology 140, 286-91.

14 CHAPTER 2

Effect of steroids and nitric oxide on pituitary hormone release in ovariectomized, peripubertal rats

15 Abstract The purpose of this study was to determine the effects of the duration of steroid depletion on the steroid-induced luteinizing hormone and prolactin surges in ovariectomized, peripubertal female rats. Additionally, the role of nitric oxide in mediating the surge responses was determined. Peripubertal (6 week old) female Sprague-Dawley rats were ovariectomized. One or 3 weeks later, animals were injected with 17 ß-estradiol (50 µg, sc) followed 48 hours later by progesterone (2.5mg, sc). Effects of NO were examined by administering l-arginine (300 mg/kg, ip). The response of ovariectomized, adult females to steroid treatment was also determined. One and three weeks after ovariectomy, steroid replacement produced an LH and prolactin surge in peripubertal animals. However, both the magnitude and duration of the LH surge was greater 3 weeks after ovariectomy. While l-arginine significantly enhanced the magnitude of the LH surge 1 week after ovariectomy, by 3 weeks l-arginine caused a decrease in the duration, but not the magnitude of the surge. In contrast, l-arginine did not affect either the magnitude or duration of the prolactin surge one week after ovariectomy, but diminished the magnitude after 3 weeks of steroid depletion. In adults, steroids induced significant increases in both LH and prolactin. These results demonstrate that there is an age-related sensitivity to NO stimulation of LH, but not prolactin secretion, which is modulated by the duration of gonadal steroid hormone depletion. The differences in the responsiveness of LH and prolactin to steroid- induced stimulation in peripubertal animals demonstrate that these hormones are regulated by NO through different mechanisms.

16 Introduction Puberty is due to a number of neural changes that occur during embryonic and prepubertal development that lead to the activation of the gonadotrophin releasing hormone (GnRH) neurons and the onset of regular, hormone cyclicity and ovulation (See Gore 2002a for review). Circulating levels of ovarian steroid hormones rise as GnRH neuronal pulse amplitude and frequency increase (Sisk, et al. 2001). The gonadal steroids, especially estrogen, affect the activity of hypothalamic neurons and the sensitivity of the anterior pituitary gland (Gore, 2001), but puberty occurs due to neuronal activation that is independent of gonadal steroids (Gore, 2002a for review). Once cyclicity is established after puberty, rats have an LH and prolactin surge during the afternoon of proestrus (Ben-Jonathan et al. 1989; Gay et al. 1970; Kalra et al. 1971, 1972). These surges can be reliably reproduced in ovariectomized females using different methods of estrogen and progesterone replacement. Steroids may be administered by sequential injections (Heimke et al. 1987, Bonavera et al. 1993) or by implanting steroid pellets (e.g. Le et al. 1997, Yen & Pan 1998), although implants produce constant high levels of steroid hormone that desensitize the pituitary gland to GnRH stimulation (for review see Gharib et al. 1990). Sequential injections of estrogen and progesterone most closely resemble the estrous cycle of the rat and produce an LH surge in mature rats from 1 day – 8 weeks after ovariectomy (Legan et al. 1973, Caligaris et al. 1974, Blake 1977, Adler et al. 1983, Clough & Rodriguez-Sierra 1983, Rubin et al. 1985, Pi 1986, Bonavera et al. 1993). Both the LH and prolactin surges are induced by estrogen and progesterone, but the hypothalamic neural mechanisms regulating these surges are different (Ben-Jonathan et al. 1989, Caligaris et al. 1974, Neill et al. 1974). Prolactin is primarily under tonic, inhibitory control from hypothalamic dopamine, but estrogen modulates prolactin secretion by acting on hypothalamic dopamine and directly on anterior pituitary lactotrophs (See Ben-Jonathan & Hnasko 2001, Freeman et al. 2000 for reviews). Increased estrogen, which stimulates the pre- ovulatory prolactin surge (Neill et al. 1971), is concentrated in dopaminergic neurons in the arcuate nucleus of the hypothalamus (Sar 1984). Furthermore, prolactin is regulated by a number of releasing factors that are also potential targets of estrogen modulation. LH release is controlled by the pulsatile secretion of hypothalamic GnRH (See Herbison & Pape, 2001 for review). Increased estrogen produces increases in GnRH pulses and LH release (see Etgen et al.

17 1999 for review). Estrogen may act directly on GnRH neurons, probably through estrogen receptor β (Hrabovszky et al. 2000) or indirectly through transynaptic mechanisms or glial cell interaction (See Herbison and Pape 2001 and Pape, 2001 for review). One potential mediator of estrogen action on the hypothalamus is nitric oxide (NO). NO is an endogenously produced gaseous neurotransmitter that is formed from the substrate L- arginine via the action of the enzyme, nitric oxide synthase (NOS) (Bredt 1999). Once formed, NO exerts its effects on target neurons by influencing the function of second messengers, such as cyclic GMP (see Dawson & Snyder 1994, Krumenacker et al. 2004 for reviews). GnRH neurons in the hypothalamus are surrounded by NOS-positive neurons, suggesting that NO regulates these neurons (Bhat et al. 1996, Bredt 1999, Grossman et al. 1994, Herbison et al. 1996). NO stimulates GnRH release (Bonavera et al. 1993) and synthesis (Wang et al. 1998) and may play a role in the induction of the steroid induced LH surge (Aguan 1996, Bonavera et al. 1994, Bonavera et al. 1993). While the role of NO in mediating the steroid-induced prolactin surge is not clear, NO stimulates prolactin release and inhibits dopaminergic neuronal activity in the median eminence (Yen & Pan 1999). Furthermore, excitatory amino acids (Abbud & Smith 1991), particularly glutamate (VanDenPol & Trombley 1993) stimulate prolactin release (see Freeman et al. 2000 for review), and excitatory amino acids induce NO release (Dhandapani & Brann 2000). The goal of this study was to examine the effects of NO supplementation and the duration of steroid depletion, induced by ovariectomy, on the steroid-induced LH and prolactin surges in peripubertal female rats. The results indicate that there is an age-related sensitivity to steroid hormone replacement and to NO stimulation that is modulated by the duration of gonadal steroid hormone depletion.

Methods & Materials

Animals and Treatments: Female rats (Ratus ratus) of the Sprague Dawley strain were purchased from Harlan Laboratories (Indianapolis, IN). All rats were ovariectomized at either 6 weeks of age, prior to vaginal opening, or as mature adults (~16 weeks of age). Animals were housed in the animal facility at Miami University on a 12L:12D cycle (lights on at 0300h). Food and water

18 were available ad libitum. All procedures were performed in accordance with the National Institutes of Health (NIH) guidelines and were approved by the Miami University Institutional Animal Care and Use Committee (IACUC). Surgical Preparation and Gonadal Steroid Treatment: Peripubertal rats (6 weeks old) were examined, and only those that did not have vaginal openings were used in these studies, i.e. those that have not yet started estrous cyclicity (Docke et al. 1974, Urbanski et al. 1987, 1998). Typically, vaginal openings occur in our animal population between 39-45 days of age. Bilateral ovariectomies were performed on all animals under isoflurane/oxygen gas anesthesia. Peripubertal animals were allowed to recover for 7 or 21 days. Adult animals were given a 21 day recovery period. We previously examined the steroid-induced LH and prolactin surges in adult animals after 7 days of steroid depletion (Brown et al. 2004). Following recovery, rats were injected with 17-ß-estradiol (50 µg/kg, sc) or sesame oil at 0700h (Day 1). The day after the estradiol injection and one day prior to their use in an experiment, indwelling jugular catheters were surgically implanted in each rat under isoflurane/oxygen gas anesthesia as previously described (Callahan et al. 2000, Jaworski-Parman et al. 1997). On the day of the experiment (Day 3), animals received progesterone (2.5 mg/kg, sc) or sesame oil at 0700h. This regimen of steroid replacement is known to induce an LH surge in mature, ovariectomized rats (Heimke et al. 1987). l-arginine Administration: In a second study, separate groups of steroid-treated, ovariectomized, peripubertal females received l-arginine (150 mg/kg, ip) or saline at 1000 or 1200 h on the day of the experiment following the protocol of Yen and Pan (1999). Steroids, sesame oil, and L- arginine were purchased from Sigma Chemical Co. (St. Louis, MO). Blood Sampling and Tissue Collection: On the day of the experiment, blood samples (0.8 ml) were withdrawn through the previously implanted jugular cannula at 0900, 1100, 1200, 1300, 1400, 1600 and 1800h. Blood volume was immediately replaced with an equal volume of sterile, heparinized (50 U/ml) saline to prevent alterations in hormone levels due to any changes in volume (Brown et al. 2004). All blood samples were kept at 4oC until the end of the experiment. Following the last blood sample, rats were sacrificed and the uterus (including the cervix) was removed from each rat and the fat was removed. Uteri were blotted on absorbent paper, and wet uterine weights were recorded. Wet uterine weights are reported as a percentage of body weight.

19 The blood was centrifuged (1000 xg) and the plasma was collected and stored at –20oC until subjected to radioimmunoassay. Radioimmunoassay (RIA): Plasma samples were assayed in duplicate for LH and PRL using reagents provided by the National Hormone and Peptide Program (NHPP), the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), and Dr. A. F. Parlow (Harbor-UCLA Research and Education Institute). Hormone levels are expressed in terms of rat PRL Reference Prep-3 or rat LH Reference Prep-3. Goat anti-rabbit gamma globulin was purchased from

Antibodies Inc. (Davis, CA). 125I-labeled PRL and 125I-labeled LH were purchased from Covance Laboratories (Vienna, VA). Plasma samples from a given experiment were assayed in a single RIA. Intraassay and interassay coefficients of variation were less than 8% and 12%, respectively. Statistical Analysis: All data were analyzed using a repeated measures ANOVA design using software from Statistical Analysis System, Inc. T tests were employed to determine whether various sample group means differed sufficiently to indicate differences in the corresponding population means. These tests were performed using the Bonferroni multiple comparison approach in order to control the overall experimental error. The overall error rate was controlled at α = 0.1 for hormone data and α = 0.05 for uterine weight. Because ANOVA designs assume that all groups have normally distributed errors with constant variance, LH values for the 6 week old animals given a three week recovery period were log transformed prior to analysis to deal with outliers and the fact that variability in this group was much larger than in the other groups.

Results Ovariectomy + EP: Basal and Peak Hormone Levels In peripubertal animals, there was a significant increase in basal levels of LH, but not PRL, by three weeks post-ovariectomy (Table 2.1). Regardless of age at the time of ovariectomy or the duration of steroid depletion, steroid replacement induced a significant LH and PRL surge in adult and peripubertal females (Table 2.1). However, the LH surge was significantly greater in the peripubertal animals after three weeks of steroid depletion. In contrast, the magnitude of the PRL surge was greatest in adult, ovariectomized animals (Table 2.1).

20 Hormone levels in peripubertal females following one week of steroid depletion When administered one week after ovariectomy, steroid hormones induced a significant increase in LH in peripubertal females and l-arginine administration significantly increased the magnitude of the LH surge (Fig 2.1). Although a PRL surge was also induced by this steroid treatment, l-arginine did not have any effect on the prolactin response (Fig 2.2). Hormone Levels in peripubertal females following three weeks of steroid depletion When administered three weeks after ovariectomy, steroid hormones induced a significant increase in the magnitude and duration of the LH surge in peripubertal females (Fig 2.3) compared to one week of steroid depletion (Fig 2.1) and to adults (Fig 2.4, Table 2.1). Administration of l-arginine did not increase the magnitude of the LH surge. In fact, the duration of the surge decreased with L-arginine supplementation (Fig 2.3). Steroid treatment also induced a PRL surge in peripubertal females (Fig 2.5) that was similar to the surge induced by steroids after one week of steroid depletion (Fig 2.2), but significantly less than the PRL surge in adults (Fig 2.6). The magnitude and duration of the PRL surge was significantly decreased by l-arginine administration. Uterine Weight Regardless of duration of steroid depletion, there was a significant increase in uterine weight following E+P treatment (Fig 2.7). However, by three weeks post-ovariectomy, the increase in uterine weight was significantly less than the increase after one week. Steroid replacement also increased uterine weight in the adult females (Fig 2.8) and the increase was similar to that observed in peripubertal females after 3 weeks of steroid depletion.

Discussion

The results of this study indicate that increases in NO, due to l-arginine administration, increased the magnitude of the steroid-induced LH surge in peripubertal, ovariectomized female rats, but the stimulatory effects of NO are related to the duration of steroid depletion. Furthermore, basal levels of LH were elevated in peripubertal females 3 weeks after ovariectomy and these animals were more responsive to steroid-induced LH secretion. One possible explanation for these results is, that as the interval between ovariectomy and steroid replacement increased, circulating levels of gonadotrophins increased due to loss of negative feedback (Gore

21 et al. 1997, Herbison 1998, Sagrillo et al. 1996). This causes the pituitary to be more responsive to GnRH stimulation (Legan et al. 1973) and results in increased LH release following steroid hormone treatment. Another possibility is that, as the animal ages, it becomes more responsive to steroid hormone stimulation. It is clear that as maturation occurs, a number of developmental changes occur that influence the activity of neural factors required for the induction of the LH surge, including maturation of the GABA (see Ojeda et al. 2003) and glutamatergic pathways, as well as NMDA receptors (Brann et al. 1997, Gore 2001, Urbanski et al. 1987, 1998). However, previous results from our laboratory indicate that age alone cannot explain this increased responsiveness to steroids (Brown et al. 2004). The same regimen of steroid hormone replacement used in this study was administered to 9 week old females one week after ovariectomy, and the steroid-induced LH response was not greater than the levels reported in this study for 6 week old, peripubertal animals (Brown et al. 2004). Therefore, it is more likely that it is the duration of the steroid depletion that alters the sensitivity of the pituitary and/or hypothalamus to steroid stimulation (Legan et al. 1973, King & Letourneau 1994), resulting in an increased LH surge. The magnitude of the steroid-induced LH surge in peripubertal animals (28.1 + 7.0 ng/ml at 1400 h) after one week of steroid depletion was similar to levels previously reported for adults (34.74 + 12.5 ng/ml at 1400h; Brown et al. 2004). L-arginine supplementation significantly increased the magnitude of the LH surge in these peripubertal, ovariectomized females. The involvement of NO in mediating the steroid-induced LH surge has already been shown in adult, ovariectomized females (Bonavera et al. 1993, Bonavera et al. 1996, Brann et al. 1997), but this is the first report that NO enhances the LH surge in peripubertal animals. However, the facilitory effects of NO are dependent on steroid exposure because after 3 weeks of steroid depletion, NO actually decreased the magnitude and duration of the LH surge. One possible explanation for the decrease in the LH response to l-arginine is that the longer duration of estrogen depletion caused a decreased sensitivity of NOS. It has been reported that estrogen increased hypothalamic NOS activity (Gouveia et al. 2004, McCann et al. 1998) and gene expression (Sahu 1998), as well as NOS activity and protein levels in the pituitary (Garrel et al. 1998). Therefore, following the longer period of steroid depletion, NOS activity and/or levels may have been reduced. A second possibility for the decreased LH response to l-arginine supplementation is that NOS levels decreased as the animals matured. An age-related decrease in hypothalamic NOS gene

22 expression has been reported, but this occurred in middle-aged rats (Sahu 1998). Therefore, it seems unlikely that the decreased LH response to l-arginine is due to an age-related decline in NOS expression because the rats used in this study were young. Steroid hormone replacement also caused a significant increase in prolactin secretion in peripubertal animals, and although the timing of the surge was similar to that seen in adult ovariectomized females, the magnitude of the surge was much lower. Neither the duration of steroid depletion, nor the administration of l-arginine increased the magnitude of the prolactin secretory response to steroid hormone administration. In fact, after three weeks of steroid depletion, l-arginine significantly decreased the magnitude and duration of the prolactin response to steroid treatment. Although NO appears to play a role in mediating the steroid-induced prolactin surge (Bonavera et al. 1994, Yen & Pan 1999), effects of increased NO levels vary depending on the site of NO action and the methods employed to produce increases in NO. For example, increased levels of NO produced by administration of an NO donor, produced a decrease in prolactin secretion from hemipituitary glands collected from male rats (Duvilanski et al. 1996), suggesting NO inhibits prolactin secretion. However, NO was essential in mediating the interleukin-1-β-induced stimulated release of prolactin in cultured pituitary cells from male rats (Brunetti et al. 1995). On the other hand, at the hypothalamic level, NO stimulated prolactin secretion in the male by inhibiting hypothalamic tyrosine hydroxylase activity (Gonzalez et al. 1998). Similarly, inhibitors of NOS synthesis prevented the estrogen-induced inhibition of dopaminergic activity in the median eminence (Yen & Pan 1999), as well as the prolactin surge (Bonavera et al. 1994) in female rats. However, l-arginine administration, which also increases NO levels, did not have any effect on either dopaminergic activity or prolactin levels in ovariectomized, steroid treated rats (Yen & Pan 1999). In our study, l-arginine was administered systemically following the protocol of Yen and Pan (1999). Our results, in agreement with Yen and Pan (1999), demonstrate that l-arginine had no effect on circulating levels of prolactin following one week of steroid depletion. Our results also confirm those of Velardez et al. (2003) who reported that increases in NO did not affect prolactin release from anterior pituitary cells collected from rats 2 weeks after ovariectomy and treated with 17-β-estradiol. After 3 weeks of steroid depletion, however, l-arginine significantly decreased the prolactin response to steroid hormone stimulation, suggesting that the inhibitory effects of NO, probably at the level of the anterior pituitary gland, were increased (Duvilanski 1996).

23 Uterine weight was used as a marker of steroid hormone action. As expected, steroid hormone replacement produced an increase in uterine weight in all treatment groups. However, while steroid replacement increased uterine weight in ovariectomized, peripubertal females, the increase was greater after only one week of steroid depletion. In addition, the magnitude of the increase in uterine weight was similar in the peripubertal and adult, ovariectomized females after 3 weeks of steroid depletion. These results indicate that, regardless of the age of the animal, the uterus loses sensitivity to steroid effects over time. We have previously reported that there is greater uterine growth when steroid replacement occurs in a cyclic, repeated pattern (Brown et al. 2004). In conclusion, these results demonstrate that there is a sensitivity to NO stimulation of LH, but not prolactin, that is modulated by the duration of gonadal steroid hormone depletion. Furthermore, three weeks of steroid depletion increased basal levels of LH, but not prolactin indicating a loss of negative feedback in the regulation of LH. The differences in the responsiveness of LH and prolactin to steroid-induced stimulation in peripubertal animals demonstrate that these hormones are regulated by NO through different mechanisms.

Acknowledgments Supported by NIH DK 54065-01 grant to P. Callahan

24 References

Bonavera J, Kalra S and Kalra P (1994) Evidence in support of nitric oxide (NO) involvement in the cyclic release of prolactin and LH surges. Brain Research 660, 175-179.

Brann D, Bhat G, Lamar C and Mahesh V (1997) Gaseous transmitters and neuroendocrine regulation. Neuroendocrinology 65, 385-395.

Bredt D (1999) Endogenous nitric oxide synthesis: biological functions and pathophysiology. Free Radical Research 31, 577-596.

Brown A, Janik JM, Murphree ES, King R and Callahan P (2004) Effects of cyclic steroid hormone replacement on prolactin and luteinizing hormone surges in female rats. Reproduction 128(3), 373-378.

25 Callahan P, Klosterman S, Prunty D, Tompkins J and Janik J (2000) Effects of immunoneutralization of endogenous opioid peptides on prolactin secretion during lactation. Neuroendocrinology 71, 268-276.

Docke F and Dorner G (1974) Oestrogen and the control of gonadotrophin secretion in the immature rat. J Endocrinology 63, 285-298.

Duvilanski B, C Zambruno, M Lasaga, D Piser, A Seilicovich (1996) Role of nitric oxide/cyclic GMP pathway in the inhibitory effect of GABA and dopamine on prolactin release. J Neuroendocinology 8, 909-913.

Gore A (2001) Gonadotropin-releasing hormone neurons, NMDA receptors, and their regulation by steroid hormones across the reproductive life cycle. Brain Research Reviews 37, 235-248.

Gore A and Roberts J (1997) Regulation of gonadotropin-releasing hormone gene expression in vivo and in vitro. Frontiers in Neuroendocrinology 18, 209-245.

Gouveia E and Franci C (2004) Involvement of serotonin 5HT1 and 5HT2 receptors and nitric oxide synthase in the medial preoptic area on gonadotropin secretion. Brain Research Bulletin 63, 243-251.

Heimke C, Schmid A, Buttner D and Ghraf R (1987) Improved model for the induction of proestrus-like gonadotropin surges in long-term ovariectomized rats. J Steroid Biochemistry 28, 357-359.

Herbison A (1998) Multimodal influence of estrogen upon gonadotropin-releasing hormone neurons. Endocrine Reviews 19, 302-330.

Jaworski-Parman R, Callahan P and Janik J (1997) Immunoneutralization of β-endorphin blocks prolactin release during suckling without affecting tuberoinfundibular dopaminergic neuronal activity in post-partum rats. Life Sciences 61, 1301-1311.

McCann S, Kimura M, Walczewska A, Karanth S, Rettori V and Yu W (1998) Hypothalamic control of FSH and LH by FSH-RF, LHRH, cytokines, leptin and nitric oxide. Neuroimmunomodulation 5, 193-202.

Ojeda S, Prevot V, Heger S, Lomniczi A, Dziedzic B and Mungenast A (2003) Glia-to-neuron signaling and the neuroendocrine control of female puberty. Annals of Medicine 35, 244-255.

Sagrillo C, Grattan D, McCarthy M and Selmanoff M (1996) Hormonal and neurotransmitter regulation of GnRH gene expression and related reproductive behaviours. Behavioral Genetics 26, 241-277.

Urbanski H and Ojeda S (1987) Neuroendocrine mechanisms controlling the onset of female puberty. Reproductive Toxicology 1, 129-138.

Urbanski H and Ojeda S (1998) Neuroendocrine mechanisms controlling the onset of female puberty. Repro Tox 1, 129-138.

27 Table 2.1. Basal and peak LH and PRL levels in female rats following ovariectomy (ng/ml).

Animal Group Basal LH Peak LH Basal PRL Peak PRL Peripubertal (1 wk; n=8) 5.8 + 1.1 28.1 + 7.0 * 24.6 + 8.0 284.2 + 16.6 * †

Peripubertal (3 wks; n=11) 18.8 + 2.8ψ 179.9 + 32.8 * † ψ 44.9 + 11.1 231.4 + 59.5 * †

Adult (1 wk; n=7) 12.6 + 1.9 34.7 + 12.6 * 23.5 + 15.7 716.4 + 278.1 *

Adult (3 wks; n=15) 17.4 + 2.1 63.1 + 14.4 * 63.2 + 21.3 1745.7 + 182.6 *

Adult females were 16 weeks old and peripubertal animals were 6 weeks old at the time of the ovariectomy. The post-ovariectomy recovery period and the number of animals in each group are in parentheses. Peak levels of LH and PRL were significantly greater than basal levels, i.e. levels at 0900 h, within the same group. The time of the steroid-induced LH peak was 1300 or 1600 h in peripubertal animals after one or three weeks of steroid depletion, respectively. In adult animals, LH levels peaked at 1400 h. PRL levels reached peak values at 1100 h in peripubertal animals, regardless of the recovery time, whereas in adult animals, PRL levels reached peak values at 1200 h. All p values < 0.006. * Significantly different from basal levels in same treatment group. † Significantly different from peak levels in adults. Ψ Significantly different from peripubertal group, 1 week after ovariectomy.

28 Figure Legends

Figure 2.1 The effect of steroid hormone (EP) replacement on LH levels in peripubertal females, 1 week post-ovariectomy. Peak LH levels were significantly increased with supplemental l-arginine (Larg). * Significantly different from baseline (0900 h), p<0.0053. † Significantly different between treatment groups, p<0.0053.

Figure 2.2 The effect of steroid hormone (EP) replacement on PRL levels in peripubertal females, 1 week post-ovariectomy. Peak PRL levels were not different between the control and l-arginine (Larg) supplemented groups. * Significantly different from baseline (basal levels, i.e. levels at 0900 h), p<0.0053.

Figure 2.3 The effect of steroid hormone (EP) replacement on LH levels in peripubertal females, 3 weeks post-ovariectomy. Administration of gonadal steroids induced a significant increase in LH levels, but l-arginine (Larg) treatment inhibited the duration of the surge. * Significantly different from baseline (0900 h), p<0.0053. † Significantly different between treatment groups, p<0.0053.

Figure 2.4 The effect of E + P administration on LH levels in adult females, 3 weeks post-ovariectomy. LH levels surged at 1400 h. * Significantly different from baseline (0900 h), p<0.0053.

29 Figure 2.5 The effect of steroid hormone (EP) replacement on PRL levels in peripubertal females, 3 weeks post-ovariectomy. PRL levels surged at 1100 h and remained significantly elevated at 1200 h. Administration of l-arginine (Larg) decreased the magnitude and duration of the PRL surge. * Significantly different from baseline (0900 h), p<0.0053. † Significantly different between treatment groups, p<0.0053.

Figure 2.6 The effect of E+P administration on PRL levels in adult females, 3 weeks post-ovariectomy. PRL levels surged at 1100 h and remained significantly elevated at 1200 h. * Significantly different from baseline (0900 h), p<0.0053.

Figure 2.7 The effect of vehicle (oil) or steroid hormone replacement, administered at 1 or 3 weeks after ovariectomy, on uterine weight as a percentage of body weight in peripubertal females. * Significantly different from controls, p<0.0056. † Significantly different from steroid-treated females after one week of steroid depletion, p<0.0056.

Figure 2.8 The effect of oil (control) or steroid hormone replacement on uterine weight, expressed as a percentage of body weight, in adult females, 3 weeks after ovariectomy. * Significantly different from controls, p<0.0056.

30 Figure 2.1

250 EP EP+Larg 200

150

† *

LH (ng/ml) LH 100 † * * 50 * * *

0 900 1100 1200 1300 1400 1600 1800 Time of Day (hr)

31 Figure 2.2

300 * EP EP + Larg * 250 * 200 * 150 * PRL (ng/ml) 100

0 900 1100 1200 1300 1400 1600 1800 Time of day (hr)

32 Figure 2.3

EP 250 EP+Larg

* 200 † *

150 * * * * *

LH (ng/ml) LH 100 † * 50

0 900 1100 1200 1300 1400 1600 1800 Time of Day (hr)

33 Figure 2.4

100

* 60

LH (ng/ml) 40

0 900 1100 1200 1300 1400 1600 1800 Time of day (hr)

34 Figure 2.5

EP 300 E/P + Larg † 250 * * 200

150 * PRL (ng/ml) 100

0 900 1100 1200 1300 1400 1600 1800 Time of Day (hr)

35 Figure 2.6

2500

2000 *

1500

PRL (ng/ml) 1000

500

0 900 1100 1200 1300 1400 1600 1800 Time (hour)

36 Figure 2.7

0.50 1 wk Oil 0.45 * 1 wk EP 0.40 3 wks Oil 0.35 0.30 3 wks EP 0.25 0.20 †* % Body Weight % Body 0.15 0.10 0.05 0.00 Treatment Groups

37 Figure 2.8

0.25 OVX + Oil OVX + E/P *

0.20

0.15

% Body Weight 0.10

0.05

0.00 Treatment

38 CHAPTER 3

Effect of steroids on prolactin release, tyrosine hydroxylase and nitric oxide synthase in young and adult rats

39 Abstract The purpose of this study was to investigate the effects of gonadal steroid hormones on anterior pituitary prolactin secretion and on hypothalamic nitric oxide (NO) and tyrosine hydroxylase (TH) levels during the prepubertal period in rats. Four week old rats were ovariectomized and, following a 21 day recovery period, were treated with oil (control) or 17-β estradiol (50 µg/kg) followed by progesterone (2.5 mg/kg) one day later. Four-week-old intact females, ovariectomized, oil and steroid-replaced females were used. Adult, ovariectomized oil and steroid replaced females were included for comparison. The effect of steroids on the prolactin secretory response on hypothalamic TH and nitric oxide synthase (NOS) expression was determined. Gonadal steroid hormones increased hypothalamic TH levels in ovariectomized, prepubertal rats, while also increasing circulating levels of prolactin (PRL). Additionally, TH levels were lower in ovariectomized, prepubertal rats compared to intact females, regardless of steroid treatment. As expected, administration of gonadal steroids caused a significant decrease in TH levels and a concomitant increase in circulating PRL levels in adults. While steroid hormone treatment produced an increase in hypothalamic NOS levels in adult, ovariectomized females, steroids did not significantly affect NOS levels in prepubertal females. The levels in the steroid-replaced, ovariectomized and intact prepubertal females were the same and, although they were higher than in the ovariectomized controls, this difference was not significant. These results indicate that gonadal steroids do influence prolactin secretion, as well as TH expression levels, in the prepubertal female rat, but the effects are different from those in adults. Furthermore, these results support the hypothesis that the increase in PRL during pubertal development is influenced by the steroid milieu, and that this increased PRL activates tuberoinfundibular dopaminergic neurons. Further studies, carefully and systematically investigating the time course of pubertal development, will help elucidate the complex mechanisms involved in the regulation of PRL release by ovarian steroids during puberty.

40 The physiological mechanisms involved in the transition to adult cyclicity in females are extremely complex, but it is clear that the hypothalamus is the site of vital neurochemical activity both during the onset of puberty and in the regulation of the adult pattern of cyclic pituitary secretions (Clough et al. 1983, Grumbach 2002). Puberty occurs due to neural changes in the brain that are independent of gonadal steroids (Gore, 2002), but at some point during development, the steroid hormones influence hypothalamic neural activity directly and indirectly (DeMaria et al. 2000, Herbison 1998). The complex interactions between the ovarian steroids and hypothalamic neuromodulators persist, and produce the cyclic pattern of anterior pituitary hormonal secretion characteristic of a mature female mammal. In the rat, this pattern of secretion includes a surge in both prolactin (PRL) and luteinizing hormone (LH) during proestrous. Although the LH and PRL surges are induced by ovarian steroids, the hypothalamic neural mechanisms regulating these surges are different (Caligaris et al. 1972, Neill et al. 1974). Estrogen produces increases in hypothalamic Gonadotropin Releasing Hormone (GnRH) pulses and, consequently, LH secretion increases (see Etgen et al. 1999 for review). Prolactin is primarily under tonic, inhibitory control from hypothalamic dopaminergic neurons (Ben- Jonathan et al. 2001) and estrogen has been localized in the nuclei of these neurons, indicating that these neurons are also sensitive to estrogen modulation (Sar 1984) A number of other neuromediators have been shown to influence hypothalamic GnRH and dopaminergic neurons. For example, norepinephrine (NE) (Adler et al. 1983, Etgen et al. 2001) glutamate (Claypool et al. 2000) and serotonin (Gouveia et al. 2004) stimulate GnRH activity and LH release, while γ-aminobutyric acid (GABA) inhibits GnRH (Feleder et al. 1999) and dopaminergic activity (Lee et al. 2001). Glutamate (Aguilar et al. 1997) and angiotensin II (Moreno et al. 2004) stimulate dopaminergic activity. The effects of nitric oxide (NO) vary depending upon its site of action, but it decrease dopamine release from the median eminence (Lafuente et al. 2004). The current study focuses on the modulatory effect of NO on PRL secretion. Nitric oxide, formed from L-arginine via the action of the enzyme nitric oxide synthase (NOS) (Bredt 1999), acts as a neuromediator in the control of GnRH and dopamine (DA) secretion (Bonavera et al. 1994, Bonavera et al. 1993, Duvilanski 1996, Lonart et al. 1994, Melis et al. 1996, Moretto et al. 1993, Rettori V 1993, Seilicovich et al. 1995, Wang et al. 1998, Yen et al. 1999). One of the three isoforms of NOS, neuronal NOS (n-NOS), is abundantly expressed in

41 the hypothalamic region, is the major NOS isoform responsible for NO production in the hypothalamus, and is known to regulate anterior pituitary secretory function (Bhat et al. 1995). The importance of n-NOS in hypothalamic activity is supported by the finding that NOS activity in the hypothalamus of endothelial NOS (e-NOS) knock-out mice is normal, but eliminated in the hypothalamus of n-NOS knockout mice (Hara et al. 1996). There is also evidence that

estrogen (E2) is an important regulator of NOS in the hypothalamus because E2 treatment up- regulates n-NOS mRNA, and staining of the NO cofactor, NADPH-diaphorase (Ceccatelli et al. 1996, Herbison et al. 1996, Okamura et al. 1995, Okamura et al. 1994). Furthermore, n-NOS mRNA and protein levels in the hypothalamus of the rat were increased on the afternoon of proestrus, providing evidence for a cyclical regulation of NO by steroids (Brann et al. 1997). Nitric oxide neurons are also present in hypothalamic nuclei important for GnRH regulation. NOS immunopositive neurons have been shown to surround hypothalamic GnRH immunopositive neurons, indicating possible contact between them (Aguan 1996, Bhat et al. 1995, Grossman et al. 1994, Herbison et al. 1996), but no NOS/GnRH double-labeled hypothalamic neurons have been detected. Furthermore, NO participates in the control of the steroid-induced LH surge and mediates the stimulatory effect of excitatory amino acids on LH and PRL surges during the estrous cycle in adult females (Andries et al. 1995, Bonavera et al. 1994, Bonavera et al. 1993). However, the role of n-NOS in the induction of the first LH and PRL surges initiating reproductive maturity is not known. Although the regulation of PRL release is complex and involves several different hypothalamic factors, hypothalamic DA is clearly the primary regulator of PRL release, providing tonic, inhibitory control of PRL secretion (Ben-Jonathan et al. 2001). Dopamine synthesis is controlled by tyrosine hydroxylase (TH), the rate-limiting enzyme that catalyzes the conversion of tyrosine to L-DOPA, the first step in catecholamine neurotransmitter production (Benavides-Piccione et al. 2003). One of the three hypothalamic, dopaminergic pathways that inhibits PRL secretion is the tuberoinfundibular dopaminergic neuronal pathway (TIDA) (Moore and Lookingland 1995). Activity of TIDA neurons decreases coincident with the onset of the proestrous surge of PRL and during the estrogen/progesterone (EP)-induced PRL surge

(DeMaria et al. 2000). TIDA neurons express E2 receptors (Hou et al. 2003), and E2 has a direct

effect on these neurons, specifically E2 decreases TH activity, TH mRNA levels, and DA metabolism in the hypothalamus (Arbogast et al. 1993, DeMaria et al. 2000).

42 In addition to DA, PRL secretion is regulated by a number of other factors, including NO. Many TH-immunopositive neurons co-express NOS (Benavides-Piccione et al. 2003) and NO has been implicated as an important mediator of DA activity and PRL secretion because it is influenced by steroid hormone levels (Grumbach 2002, Kriegsfeld et al. 2002, Moreno et al. 2004, Velardez et al. 2003). It is not clear when the hypothalamic dopaminergic neurons develop sensitivity to NO, but the steroid-induced PRL surge in 6 week old, peripubertal, ovariectomized female rats was significantly less than that produced in adults, even when the peripubertal animals received l-arginine supplementation to increase endogenous NO levels (Russell et al. 2004, in review). The aim of this work was to investigate the effects of gonadal steroid hormones on anterior pituitary PRL secretion and on hypothalamic NO and TH levels during the prepubertal period in rats. Four-week-old rats were used in this study because the neural processes required for puberty have been initiated (see Gore 2002 for review). Therefore, we hypothesized that these animals would be sensitive to steroid hormone treatment. We predicted that circulating levels of PRL would increase in animals that received steroid treatment and that this effect would be mediated by a steroid-induced decrease in hypothalamic TH levels and an increase in hypothalamic NOS levels.

Methods & Materials

Animals and Treatments: Female, Fisher rats were purchased from Harlan Laboratories (Indianapolis, IN). All rats were ovariectomized at either 4 weeks of age and prior to vaginal opening (prepubertal) or at 12 weeks of age, i.e. sexually mature. All animals were housed in the animal facility at Miami University on a 12L:12D cycle (lights on at 0300h). Food and water were available ad libitum. All procedures were performed in accordance with the National Institutes of Health (NIH) guidelines and were approved by the Miami University Institutional Animal Care and Use Committee (IACUC).

Surgical Preparation and Gonadal Steroid Treatment: Bilateral ovariectomies were performed under isoflurane/oxygen gas anesthesia and rats were allowed to recover for 21 days. Following the recovery period, rats were injected with 17-ß-estradiol (E) (50 µg/kg, sc) or sesame oil at

43 0700h (Day 1). On the day of the experiment (Day 3), animals received progesterone (P) (2.5 mg/kg, sc) or sesame oil at 0700h. This regimen of steroid replacement is known to induce an LH surge in mature, ovariectomized rats (Heimke et al. 1987).

Blood Sampling and Tissue Collection: On the day of the experiment, animals were sacrificed at 1200 or 1400 h. This time course was designed to ensure that blood samples would be collected during the time of the LH and PRL surge (Bonavera et al. 1994). The samples from both of these times were pooled to represent the response during the surge. Trunk blood was collected, and the hypothalamus, cortex (positive control), kidney (negative control) and uterus (including the cervix) were removed from each rat. All blood samples were stored at 4oC until centrifuged (1000xg, 5 minutes). The plasma was collected and stored at –20oC until a PRL radioimmunoassay was performed. Whole hypothalamus, and equal amounts of cortex and kidney protein were placed in ice cold HEPES buffer containing PMSF (Keilhoff et al. 1996) and sonicated. Protein content was determined in a 10 µl aliquot of the sonicated tissue by BCA protein assay (Pierce, Rockford, IL). Tissue was stored at a final concentration of 3 µg/µl at – 20oC until subjected to Western Blot analysis. Uteri were blotted on absorbent paper, and wet uterine weights were recorded. Wet uterine weights are reported as a percentage of body weight.

Western Blot Protein Analysis: The sonicated tissue samples, diluted to 5, 10 or 20 µg of protein were solubilized in Laemmli sample buffer (Laemmli 1970). Protein and prestained molecular weight markers (BioRad, Hercules, CA) were separated by electrophoresis on an 8% SDS polyacrylamide gel (PAGE). Proteins were transferred onto PVDF membrane at 4oC using a total of ~2,000 mAmp of current. To check transfer efficiency, proteins on the gels were visualized with Coumassie Stain and proteins on the membrane were visualized by reversible staining with Ponceau Stain (0.5% in 1% acetic acid). After blocking for 2 hrs in 8% milk in Tris Buffered Saline/Tween (TBST), the membranes were incubated for 2h at room temperature with 1:2500 monoclonal mouse anti-neuronal nitric oxide synthase (nNOS) (BD Biosciences, Transduction Labs, Hercules, CA, USA), 1:40,000 monoclonal mouse pan-TH (BD Biosciences, Transduction Labs, Hercules, CA, USA) and 1:100,000 monoclonal mouse anti-Actin (Chemicon, Temecula, CA, USA). Actin served as an internal control protein. After washing four times with TBST, the membranes were incubated for 1.5 h at room temperature with 1:2500

44 peroxide-coupled conjugated goat anti-mouse immunoglobulin G (Chemicon, Temecula, CA, USA). After washing, bound antibodies were visualized by enhanced chemiluminescence using SuperSignal West Pico Chemiluminescent kits (Pierce, Rockford, IL). The films were scanned using Adobe Photoshop 6.0 and each band density was determined using ImageQuant software (Amersham Biosciences, Piscataway, NJ).

Radioimmunoassay (RIA): Plasma samples were assayed in duplicate for PRL using reagents provided by the National Hormone and Peptide Program (NHPP), the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDKD), and Dr. A. F. Parlow (Harbor-UCLA Research and Education Institute). Hormone levels are expressed in terms of rat PRL Reference Prep 3 (RP3). Goat anti-rabbit gamma globulin was purchased from Antibodies Inc. (Davis, 125 CA). I-labeled PRL was purchased from Covance Laboratories (Vienna, VA). Intraassay and interassay coefficients of variation were less than 5% and 8%, respectively.

Statistical Analysis Band density of the 5, 10 and 20 µg TH , NOS and Actin tissue samples was determined as band vol/µg of protein. Band density was plotted against protein concentration and a linear regression analysis was performed. Only samples resulting in an R2 value of 0.8 or greater were analyzed for statistical significance. The average TH and NOS band density was divided by mean actin level in the same sample to calculate NOS and TH ratios. These ratios were analyzed using a 2 sample t-test to detect any significant differences between steroid-treated and control rats. Overall error rates were controlled at α=0.05.

45 Results

Prolactin Response Loss of steroid hormones resulted in decreased PRL levels in animals ovariectomized at 4 (Fig 3.1a) and 12 weeks of age (Fig. 3.1b) Steroid replacement in prepubertal animals produced levels that were similar to those in intact, female rats at the same age (Fig 3.1a), but these levels were 6 times lower than the steroid-induced PRL levels in adults (Fig 3.1b).

Tyrosine Hydroxylase (TH) Levels Steroid treatment produced a significant increase in TH levels in rats ovariectomized at 4 weeks of age, but this was still significantly less than the amount detected in intact females at the same age (Fig 3.2a). In contrast, steroid replacement induced a significant decrease in hypothalamic TH levels in adult females (Fig 3.2b), which is consistent with the steroid-induced PRL increase observed in these animals. A sample western blot for TH is shown in Fig 3.3.

Nitric Oxide Synthase (NOS) Levels Steroid treatment did not significantly affect NOS levels in prepubertal ovariectomized female rats (p=0.1), even though NOS levels in steroid-treated and intact animals were almost two fold higher than those in the oil treated controls (Fig 3.4a). In fact, NOS levels were nearly identical between the intact and EP groups. In adults, EP induced a significant increase in NOS expression compared to ovariectomized controls (Fig 3.4b). A sample western blot of NOS is shown in Fig 3.3.

Uterine Weight In prepubertal females, steroid hormone replacement induced a three-fold increase in wet uterine weight compared to either oil-treated controls or intact females (Fig 3.5a). Steroid hormone replacement produced an even greater response in adult females; uterine weight increased approximately 6 fold (Fig 3.5b).

46 Discussion The results of this study demonstrate that gonadal steroids produce a significant increase in PRL levels in ovariectomized, prepubertal female rats. However, although the PRL secretory response to gonadal steroids reached the same levels as those in the intact, prepubertal females, they were significantly less than those produced in the adult. Furthermore, the steroid-induced PRL response in prepubertal females was approximately 3 fold lower than levels induced by steroid replacement in peripubertal, ovariectomized rats, i.e. rats ovariectomized at 6 weeks of age (Russell et al. 2004 in review). In contrast to the steroid-induced decrease in TH levels in adult, ovariectomized females, TH levels in the young, prepubertal animals were significantly increased by gonadal steroid replacement, but were still significantly lower than intact, prepubertal females. In addition, steroids induced a significant increase in NOS levels in adult, ovariectomized females only. While NOS levels in steroid treated and intact, prepubertal females were greater than in ovariectomized controls, the increase was not significant. Clearly the hypothalamic neural mechanisms that regulate PRL secretion and/or the anterior pituitary gland are sensitive to steroid modulation in prepubertal animals, but are not sufficiently sensitive to produce adult PRL surges typical of adults. Since the primary regulator of PRL secretion is inhibition from hypothalamic DA (Ben- Jonathan et al. 2001), we examined the effects of steroid hormone replacement on TH levels. As expected, in adults, administration of gonadal steroids caused a significant decrease in TH levels (DeMaria et al. 2000) which is consistent with the steroid-induced increase in PRL. However, the effects of estrogen on PRL secretion are complex and involve actions at both the anterior pituitary and hypothalamic levels (Ben-Jonathan and Hnasko 2001). In adult females, estrogen induces an initial decrease in hypothalamic dopamine and thus produces a PRL surge (Ben- Jonathan and Hnasko 2001). This increase in PRL, however, subsequently activates dopaminergic neurons via feedback effects (Ben-Jonathan and Hnasko 2001) and leads to increased levels of TH protein and mRNA, as well as increased TH activity (Arbogast and Voogt 1991). The overall result of this TIDA neuronal activation is that PRL levels return to basal values over time; typically, this occurs after at least 4 hours of hyperprolactinemia (Demarest et al. 1984). Our results in adult females are consistent with this previous work. In contrast to the adult response, steroid hormones actually increased hypothalamic TH levels in ovariectomized, prepubertal rats, while also increasing circulating levels of PRL.

47 Interestingly, TH levels were lower than in intact, prepubertal females, regardless of steroid treatment, but PRL levels were the same in intact and steroid-replaced, ovariectomized females. These results suggest that, although the dopaminergic neurons seemed to respond to gonadal steroids, the neural mechanisms that regulate PRL secretion in the adult are not functioning yet in the prepubertal animal. While it was surprising to find increases in both TH and PRL levels at the same time, it is important to remember that the more dynamic secretory patterns of PRL regulation, typical of adult, cycling females, do not occur in these prepubertal animals. Furthermore, in addition to inhibition by dopamine, PRL secretion is controlled by a number of other neuroregulators (see Freeman et al. 2000 for review). Also, Shieh and Pan (1999) reported that both PRL levels and basal TIDA neuronal activity gradually increased from the neonatal to pubertal stages in rats. These investigators suggested that the increased PRL activates TIDA neurons and drives them to maturation during pubertal development (Shieh et al. 1999). Our results support this hypothesis and further suggest that the rise in PRL levels that occur as the animal proceeds into puberty is influenced by the steroid milieu. This is consistent with the idea that, even in prepubertal animals, the pituitary is capable of responding to stimulation, and of functioning as it does in the adult (Debeljuk et al. 1972). Another possible explanation for the increased TH levels in the presence of high circulating levels of PRL in prepubertal rats is that PRL may stimulate TH in other hypothalamic neuronal systems, i.e., in the tuberohypophyseal dopaminergic (THDA) or paraventricular hypophyseal dopaminergic (PHDA) pathways (DeMaria et al. 1999). NOS levels were significantly increased by gonadal steroid replacement in adult, ovariectomized female rats. These results are consistent with previous reports that NOS protein and mRNA levels increased in the hypothalamus during the afternoon of proestrus (Brann 1997) and after administration of E2 to adult rats (Okamura et al. 1994, 1995, Ceccateli et al. 1996). While NOS levels in steroid-treated and intact, prepubertal females were greater than in ovariectomized controls, this increase was not significant. Clearly, the NOS system plays a role in pubertal development because when NO production was decreased pharmacologically with NOS inhibitors, puberty was delayed (Pinilla et al. 1998). In addition, the number of n-NOS immunopositive neurons in the hypothalamus in prepubertal animals is similar to that in adults, indicating a sufficient level of expression (Gorbatyuk et al. 1997), but the neural mechanisms involved in pubertal development occur independently of steroid hormone modulation (Gore

48 2002). The pituitary gland from prepubertal animals, however, is capable of responding to steroid and hypothalamic control in the same way as pituitaries from adult, sexually mature animals. Therefore, it is likely that steroid modulation of hypothalamic NOS signaling is not yet fully developed in these prepubertal animals. The stimulatory effect of estrogen on uterine size has long been a standard used to confirm that gonadal steroid treatment was effective (Ojeda et al. 1976). Steroid hormone replacement induced a significant increase in wet uterine weight in both prepubertal and adult females. In prepubertal females, this increased uterine weight was significantly greater than either the oil treated controls or intact females. The uterus in the prepubertal females was very sensitive to steroid stimulation; steroid hormone replacement produced a 5 fold increase in uterine weight. Interestingly, the uterine weight in intact, prepubertal females was not different than ovariectomized controls. These results indicate that the low levels of ovarian steroids, typical of prepubertal females, were not yet sufficient to stimulate uterine growth. In adults, as expected, steroid hormone replacement significantly increased uterine weight by approximately 7%, demonstrating that uterine tissue serves as a good indicator of estrogen action. In summary, this is the first study to investigate the role of gonadal steroids on the regulation of PRL secretion in prepubertal females. These results indicate that gonadal steroids do influence prolactin secretion, as well as TH expression levels, but the effects are different from those in adults. When gonadal steroids are depleted by ovariectomy, circulating PRL levels and hypothalamic TH expression levels decrease, indicating a dependence on ovarian steroid hormones by the late prepubertal stage. While steroid hormone treatment produced an increase in NOS levels in adult, ovariectomized females, steroids did not significantly affect NOS levels in prepubertal females. These results support the concept that there are several factors involved in mediating the steroid induced PRL surge that initiates reproductive maturity. Further studies that carefully and systematically investigate the time course of pubertal development will help elucidate the complex mechanisms involved in the regulation of PRL release by ovarian steroids during puberty.

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57 Figure Legends

Figure 3.1a PRL levels in intact (n=6), ovariectomized, oil-treated controls (OVX, n=4) and ovariectomized, steroid-treated (EP, n=5) prepubertal female rats. Blood samples were collected at either 1200 or 1400 h, times at which PRL levels increase following administration of gonadal steroids in adult, ovariectomized females. Steroid treatment induced a significant rise in PRL levels, increasing concentrations to the levels in intact females. Values are means + SEM. * Significantly different from ovariectomized controls, p< 0.016.

Figure 3.1b PRL levels in ovariectomized, oil-treated controls (OVX, n=7) and ovariectomized, steroid- treated (EP, n=6) adult female rats. Blood samples were collected at either 1200 or 1400 h, times at which PRL levels increase following administration of gonadal steroids in adult, ovariectomized females. These samples were then pooled and analyzed. Steroid treatment induced a significant increase in PRL levels. Values are means + SEM. * Significantly different from ovariectomized controls, p = 0.0017.

Figure 3.2a Hypothalamic TH levels, expressed as a ratio of total actin, in intact (n=6), ovariectomized, oil- treated controls (OVX, n=4) and ovariectomized, steroid-treated (EP, n=4) prepubertal female rats. Levels were determined at either 1200 or 1400 h and these samples were then pooled and analyzed. Steroid treatment induced a significant rise in TH levels compared to ovariectomized controls, but these were still significantly less than the levels in intact females. Values are means + SEM. Means with different letters are significantly different, p<0.016.

58 Figure 3.2b Hypothalamic TH levels, expressed as a ratio of total actin, in ovariectomized, oil-treated controls (OVX, n=4) and ovariectomized, steroid-treated (EP, n=4) adult female rats. Levels were determined at either 1200 or 1400 h, and these samples were then pooled and analyzed. Steroid treatment induced a significant decrease in TH levels compared to ovariectomized controls. Values are means + SEM. Significantly different from ovariectomized, control group, p=0.0006.

Figure 3.3 Representative western blot of hypothalamus from 4 and 12 week old ovariectomized (OVX) and ovariectomized, steroid treated (OVX+EP) animals sacrificed at the time of the PRL surge. Representative bands for NOS (155 kD), TH (58 kD), Actin (42 kD) and the molecular weight markers are as indicated on the figure.

Figure 3.4a Hypothalamic NOS levels, expressed as a ratio of total actin, in intact (n=6), ovariectomized, oil- treated controls (OVX, n=4) and ovariectomized, steroid-treated (EP, n=4) prepubertal female rats. Levels were determined at either 1200 or 1400 h, and these samples were then pooled and analyzed. NOS levels in intact and steroid treated animals were similar, and were greater than levels in ovariectomized controls, but these differences were not significant. Values are means + SEM.

Figure 3.4b Hypothalamic NOS levels, expressed as a ratio of total actin, in ovariectomized, oil-treated controls (OVX, n=4) and ovariectomized, steroid-treated (EP, n=4) adult female rats. Levels were determined at either 1200 or 1400 h, and these samples were then pooled and analyzed. Steroid treatment induced a significant increase in NOS levels compared to ovariectomized controls. Values are means + SEM. * Significantly different from ovariectomized, control group, p=0.003.

59 Figure 3.5a Uterine weight, expressed as a percent of body weight, in intact (n=6), ovariectomized, oil- treated controls (OVX, n=6) and ovariectomized, steroid-treated (EP, n=6) prepubertal female rats. Uterine weight was increased approximately 5 fold in steroid-treated females compared to oil-treated, ovariectomized controls and intact females. Values are means + SEM. *Significantly different from ovariectomized controls and intact females p< 0.016.

Figure 3.5b Uterine weight, expressed as a percent of body weight, in ovariectomized, oil-treated controls (OVX, n=6) and ovariectomized, steroid-treated (EP, n=6) adult female rats. Uterine weight was increased approximately 7 fold in steroid-treated females compared to oil-treated, ovariectomized controls. Values are means + SEM. * Significantly different from ovariectomized controls and intact females, p=0.0007.

60 Figure 3.1a

PRL Levels in 4 wk old rats

OVX 500 EP 450 Intact 400

350

300

250

200 PRL (ng/ml)

150 ** 100

0 Treatment Group

61 Figure 3.1b

PRL Levels in 12 wk old Rats

OVX 500 EP 450 *

400

350

300

250

200 PRL (ng/ml) 150

100

0 Treatment Group

62 Figure 3.2a

TH Expression in 4 wk old Rats OVX 1.20 EP Intact c 1.00

0.80

0.60 b

TH/Actin Ratio 0.40 a

0.20

0.00 Treatment Group

63 Figure 3.2b

TH Expression in 12 wk old Rats

1.20 OVX EP

1.00

0.80 *

0.60

TH/Actin Ratio 0.40

0.20

0.00 Treatment Group

64 Figure 3.3

N 5 10 20 M 5 10 20 P N 5 10 20 M 5 10 20 P µg of protein

NOS 155kD

TH 58kD

Actin 42kD

OVX OVX+EP 4 wk old 12 wk old 4 wk old 12 wk old

Fig 3 Representative Western Blot of NOS, TH and Actin 4wk old and 12 wk old OVX and OVX+EP animals N = negative control (kidney) P = positive control (cortex) M = Molecular marker

65 Figure 3.4a

NOS Expression in 4 wk old Rats

OVX 0.90 EP 0.80 Intact 0.70

0.60

0.50

0.40

NOS/Actin RatioNOS/Actin 0.30

0.20

0.10

0.00 Treatment Groups

66 Figure 3.4b

NOS Expression in 12 wk old Rats

OVX 0.90 EP 0.80 *

0.70

0.60

0.50

0.40

NOS/Actin Ratio 0.30

0.20

0.10

0.00 Treatment Groups

67 Figure 3.5a

Uterine Weight in 4 wk old Rats

OVX 0.18 EP 0.16 * Intact

0.14

0.12

0.10

0.08

0.06

0.04 Uterine Wt as % of Body Weight Body % of as Wt Uterine 0.02

0.00 Treatment Group

68 Figure 3.5b

Uterine Weight of 12 wk old Rats

OVX 0.18 EP 0.16

0.14

0.12

0.10 * 0.08

0.06

0.04 Uterine Wt as % of Body Weight Body % of as Wt Uterine 0.02

0.00 Treatment Group

69 CHAPTER 4

General Discussion

The focus of these studies was on the effect of ovarian steroids (estrogen and progesterone) on anterior pituitary hormone secretion during pubertal development in the female rat. The major focus was on the regulation of PRL secretion, but LH responses were also investigated. Previous research from our laboratory demonstrated that 6 week old rats may be in puberty, since some display vaginal openings, an indication of ovulation (Docke et al. 1974, Urbanski et al. 1987, 1998). In these 6-week old, peripubertal females, it was possible to stimulate both LH and PRL release when steroids were administered to ovariectomized females, but the magnitude of the secretion was much lower than the response produced by steroid treatment in ovariectomized adults (Brown et al. 2004). In order to examine mechanisms mediating pituitary function during pubertal development, I performed studies that examined the effects of NO on steroid-induced changes in LH and PRL release in peripubertal, ovariectomized female rats. These rats did not have vaginal openings, which indicates they had not initiated estrous cyclicity (Docke et al. 1974, Urbanski et al. 1987, 1998). However, by 6 weeks, the postnatal, developmental changes that occur in the brain allowing puberty to occur have taken place (Gore 2002). I then further examined the mechanisms of action of steroid hormones in prepubertal (4-week old) females. At 4 weeks, these animals are in an earlier stage of pubertal development, but the neural changes required for puberty are nearly completed (Gore 2002). In chapter 2, I examined the effects of the duration of steroid depletion, and of NO supplementation, on steroid-induced LH and PRL secretions in ovariectomized, peripubertal female rats. These animals were in puberty, but the first ovulation had not yet occurred. The results of the study indicated that there was an age-related sensitivity to steroid hormone replacement and to NO stimulation that was modulated by the duration of the gonadal steroid hormone depletion. Steroids induced a robust LH response during puberty that exceeds adult levels. The involvement of NO in mediating the steroid-induced LH surge in adults has already been shown in adult, ovariectomized females (Bonavera et al. 1993, Bonavera et al. 1996, Brann et al. 1997), but this is the first report to demonstrate that NO enhances the LH surge in peripubertal animals. However, the facilitory effects of NO were dependent on the length of steroid depletion, because NO actually decreased the magnitude of the LH surge following three

70 weeks of steroid depletion. This decrease in the LH response may be due to decreased sensitivity or activity of NOS in the absence of ovarian steroids. Another important finding from Chapter 2 is that during puberty, neither the duration of steroid depletion, nor the administration of L-arginine increased the magnitude of the prolactin secretory response to steroid hormone administration. NO has been shown to modulate PRL release in rats by both stimulating and inhibiting it, depending on the site of NO action and the methods employed to produce increases in NO (Bonavera et al. 1994, Brunetti et al. 1995, Duvilanski 1996, Gonzalez et al. 1999, Yen et al. 1999). During puberty, a longer period of steroid depletion resulted in an inhibitory effect of NO on PRL release. Therefore, although no stimulatory effects of NO on PRL secretion were detected, when steroid levels remained depleted for three weeks, the prolactin response to subsequent steroid replacement decreased. Taken together, these results lend additional support to the idea that the regulation of PRL and LH is mediated by different neural mechanisms. Because puberty is a dynamic period of development that lasts for several weeks, it is impossible to know if each rat in the 6-week old age group is at the same level of reproductive maturity. Even though the peripubertal rats used in Chapter 2 did not have vaginal openings, the stage of follicular development within the ovaries was unknown. Some animals at this age have begun hormonal cyclicity, have mature follicles, but have not ovulated, and therefore have closed vaginas. Others have not begun hormone cyclicity, and the ovarian follicles are still immature. In order to answer questions regarding the effects of estrogen and progesterone on the PRL secretory response during development, it was prudent to minimize variability as much as possible. This was accomplished by using younger animals. Also, I focused on the development of the regulatory mechanisms controlling PRL secretion, since little is known regarding PRL regulation during puberty. In Chapter 3, I used the Fisher strain of rat because they are more sensitive to estrogen modulation. Also, the younger four-week old rats are definitely not cycling, have no maturing ovarian follicles, and have closed vaginas. They are clearly not in puberty, but most of the neural development required for puberty to occur, has taken place in the brain (Gore 2002). The aim of this work was to determine if PRL secretion was sensitive to ovarian steroid hormones in these young, prepubertal females, and to investigate the possible modulation of hypothalamic NOS and tyrosine hydroxylase (TH) by the steroids. The results of the study indicate that

71 steroids do influence hypothalamic TH and NOS expression as well as prolactin secretion during juvenile development. In the absence of steroids, NOS, TH and PRL levels were reduced. With steroid replacement, NOS and PRL returned to the levels detected in intact, prepubertal females. While TH and PRL levels were inversely related in adults, in prepubertal females, steroid hormones increased the levels of both TH and PRL. These results indicate that dopaminergic inhibition, which is a primary regulator of PRL release in adults (Ben-Jonathan and Hnasko 2001) is not functioning the same way in the prepubertal period. These results support the concept that there are several neurochemicals involved in mediating the steroid induced PRL surge that initiates reproductive maturity. Overall, this research provides insight into the mechanisms of steroidal involvement in the onset of reproductive function. It is clear that dopamine and nitric oxide play important roles in mediating pituitary hormone function in both young animals and in adults. The current study provides the first information on the responses of hypothalamic TH, NOS and pituitary prolactin to ovarian steroids in prepubertal and peripubertal rats. Future studies that closely investigate the time course of the development of hypothalamic sensitivity to ovarian steroids will further help elucidate the mechanisms involved in the onset of puberty and regular cyclicity.

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