Chapter 2 - Elsevier Store

HOBB: 00002 2 Feminine Reproductive Behavior and Physiology in Rodents: Integration of Hormonal, Behavioral, and Environmental Influences* J D Blaustein, University of Massachusetts, Amherst, MA, USA ß 2009 Elsevier Inc. All rights reserved.

Chapter Outline

2.4.1 2.4.2 2.4.2.1 2.4.2.2 2.4.3 2.5 2.5.1 2.5.2 2.5.3 2.6 2.6.1 2.6.2 2.6.3 2.6.4

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Introduction: Approaches to the Study of Feminine Sexual Behavior Elements of Feminine Sexual Behavior Copulatory Behaviors Paracopulatory Behaviors Progestative Behaviors Sexual Motivation Mice: A Different Pattern Influence of Mating Stimulation on Long-Term and Short-Term Changes in Sexual Behavior and Neurons Vaginocervical Stimulation Short-Term Effects of Mating Stimulation Longer-Latency and Longer-Duration Enhancement of Sexual Responding by Mating Stimuli Longer-Latency Inhibition of Sexual Behavior by Mating Stimuli: Heat Abbreviation Hormonal Regulation of Steroid Hormone Receptors and Feminine Sexual Behavior Estrogen Receptors Progestin Receptors PRs and facilitation of feminine sexual behavior Downregulation of PRs leads to estrous termination and the refractory period Membrane Receptors and Sexual Behavior Crosstalk between Neurotransmitters and Steroid Hormone Receptors Neurotransmitters Influence Concentrations of ERs and PRs Ligand-Independent Activation of PRs Enhancement of Sexual Behavior by Mating: Role of Ligand-Independent Activation Mating-Induced Changes in Neuronal Activity and Response Neuroendocrine Responses to Sexual Behavior and Mating-Related Stimulation Mating-Induced Secretion of LH Mating-Induced Prolactin Secretion Mating-Induced Changes in Oxytocin Levels and Effects of Peripheral Oxytocin on Sexual Behavior Structural Changes Induced by Mating Stimulation Overview

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*This review is dedicated to Dr. Mary Erskine (1946–2007). Mary was a co-author on the first edition of this review, but met an untimely death between publication of the first and second edition. Although Mary could not contribute directly to this chapter, her contributions to the first edition, as well as to the field, were incalculable. Our review in the first edition of this volume was a true team effort with each author bringing a unique perspective to this problem. Mary spent most of her scientific career doing integrative studies aimed at understanding the neuroendocrine underpinnings and outcomes of sexual behaviors from a wide variety of perspectives. Many of the ideas in this chapter are the result of her research and her thinking. Mary is missed by her friends, trainees, and colleagues, but she will live on through her many scientific contributions and fond memories.

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2.1 Introduction: Approaches to the Study of Feminine Sexual Behavior

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A major challenge to studies of the neural and endocrine regulation of reproductive behavior and of the influence of behavior on reproductive physiology is the need to integrate multiple levels of analyses in order to understand the anatomical, cellular, and physiological processes involved. At the systems level, we can identify the behaviors, learn when and under which conditions they are expressed, and study how environmental and social factors alter their expression. Likewise, we can study the influences that particular types of stimulation received during mating has on reproductive physiology. Hypotheses about brain–behavior relationships can in turn be tested at the cellular and molecular levels. At the cellular and subcellular level, mechanistic studies of hormones and behavior can be carried out using biochemical assays for measures of metabolic and enzymatic activity. In addition, molecular mechanisms can be explored as can mechanisms of intracellular signal transduction. Pharmacological and hormonal manipulations can be used to evaluate the importance of particular neurotransmitters and hormones in the normal and abnormal functions of particular cellular systems or neural circuits. Results of experiments using each of these approaches must be

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copulatory behaviors Those behaviors which result in successful transfer of sperm from the male to the female, used instead of receptivity, because it connotes an active participation by the female, and because the same term is applied to the active behaviors shown by males. Lordosis is a sexually receptive behavior. estradiol The predominant steroid hormone (usually estradiol-17b, but the estradiol-17a isomer is also active in some systems) belonging to the class of hormones, estrogens, circulating in vertebrates. Although estradiol is sometimes called estrogen, the two terms should not be confused. estrogens A class of steroid hormones classified either by structure, by binding properties, or by their influence on particular responses. Fos Protein product of the immediate early gene, c-fos. Immunocytochemistry for the Fos protein and other protein products of immediate early genes has been used for identifying of pathways functional in a particular system. paracopulatory behaviors Species-typical behaviors displayed by females, which arouse males and stimulate them to mount. These behaviors have sometimes been termed proceptive, precopulatory, or solicitational behaviors. progestative behaviors Those behaviors that occur throughout mating, which maximize the likelihood that pregnancy will occur. progesterone The predominant steroid hormone of the class, progestins or gestagens, circulating in vertebrates. progestins A class of steroid hormones classified either by structure, by binding properties, or by their influence on particular responses. (Also called gestagens or progestagens.) pseudopregnancy A physiological condition that resembles pregnancy, which is initiated by twice daily surges of prolactin, resulting in rescue of the corpus luteum. It can result from primarily intromissive stimulation received during copulation with an infertile male or by experimenter-applied vaginocervical stimulation (VCS).

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vaginocervical stimulation (VCS) This is a reproductively relevant stimulus that is an important component of mating stimulation received by females. It can be provided by males during intromissions or ejaculations, or it can be provided by an experimenter with a glass or plastic rod. It should be noted that the experimenter-applied stimulation is primarily stimulation to the uterine cervix. ventromedial nucleus of the hypothalamus (VMN) This is the Nissl-substance-defined cluster of cells within the ventromedial hypothalamus. When authors are specific that the nucleus was studied, the term ventromedial nucleus (VMN) is used by this author. When the authors are not specific, and seem to be referring to the general area, ventromedial hypothalamus (VMH) or ventromedial hypothalamic area is used.

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Sexual behavior in female rodents includes postural stances and adjustments required for vaginal intromission by males and additional behaviors that modulate the occurrence and timing of sexual behavior and ensure successful transfer of sperm from the male to the female reproductive tract. The former behaviors are relatively stereotyped and reflexive, whereas the latter are more complex behaviors and are integral to the initiation and sequencing of behavioral events during copulation. The appropriate measure of sexual behavior chosen for any given experiment depends upon the questions being asked. If the question focuses on whether or not a particular animal is displaying estrous behavior, a simple measure of lordosis suffices. On the other hand, if the question is one of sexual readiness and the female’s motivation to mate, then a composite of the several solicitational behaviors displayed by females or tests for sexual motivation can be used. If the goal is to examine the effects of female sexual expression on neuroendocrine function and reproductive success, behaviors involved in the induction of pregnancy and facilitation of sperm transport can be studied. Studies of rodent feminine sexual behavior relied historically on the lordosis reflex as a measure of sexual behavior, and this has been quite useful in answering questions relating to the neuroendocrine bases of a relatively simple behavior. The study of the lordosis response allows for reliable assessment of the female’s responsiveness to the mating stimuli provided by the male. Likewise, it allows the study of the cellular and molecular mechanisms that underlie the expression of a relatively

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2.2 Elements of Feminine Sexual Behavior

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simple behavior and how these factors are integrated in the moment-to-moment changes in behavioral expression. The expression of lordosis can be a very useful model for studying the factors that increase or decrease the likelihood of a particular behavior. Beach (1976) proposed that female sexual behavior comprised three basic elements: receptivity, proceptivity, and attractivity. Beach acknowledged the multiple components of feminine sexual behavior and the key role that sequential interactions between partners play in the sequencing of sexual behavior. Beach’s terms have provided a structural framework for studying female sexual behavior. More recently, however, recognition of additional behavioral elements of sexual behavior, and the recognition that females are active rather than passive participants in sexual interactions has called for a revision. In the first edition of this volume, Blaustein and Erskine (2002) presented three functional components of female sexual behavior which are useful in studying the neural and endocrine regulation of female sexual responsiveness.

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integrated to achieve a comprehensive understanding of how the nervous system modifies the expression of a behavior and how that behavioral expression then alters reproductive physiology. Feminine sexual behavior, which is ultimately regulated by the brain, is influenced by stimuli from the environment, including genitosensory stimulation from mating. These afferent influences are conveyed to the brain via afferent neuronal signals. This chapter is an integration of the ways in which the social environment (mating stimulation in this case) does, and in some cases may, influence the brain, behavior, and reproductive physiology.

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Copulatory Behaviors

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The term, copulatory behaviors, those behaviors which result in successful transfer of sperm from the male to the female, is used instead of receptivity, because it connotes an active participation by females, and because the same term is applied to the active behaviors shown by males. In response to flank and perineal somatosensory stimulation received from males during copulatory mounts, females of many species, including rats, display the lordosis posture, which positions the female genitals to allow penile intromission by the male. Lordosis involves the female becoming immobile, extension of the rear legs, dorsiflexion of the spine, elevation of the head, and tail deviation (Pfaff et al., 1973). Additional postural adjustments occur during the expression of lordosis and are required to facilitate proper orientation and penile insertion by the male. Lordosis has been used as the dependent measure in the vast majority of mechanistic and neuroanatomical studies of feminine sexual behavior. Both the occurrence and magnitude of spinal dorsiflexion have been used as measures of female sexual responsiveness. The proportion of lordosis responses observed in response to a given number of mounts from the male expressed as a percentage (lordosis quotient; Whalen, 1974) or as a ratio (lordosis to mount ratio) is a measure of

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used to facilitate sexual behavior, and increasing doses of progesterone allow for lower priming doses of estradiol (Whalen, 1974). Often after exposure to progesterone, rats (Blaustein and Wade, 1977b; Morin, 1977; Powers and Moreines, 1976), hamsters (Carter et al., 1976), guinea pigs (Dempsey et al., 1936; Goy et al., 1966; Zucker, 1966, 1968), and mice (Edwards et al., 1968) become refractory to further stimulation of sexual behavior by either progesterone alone or, in some cases, estradiol and progesterone. Although progesterone desensitizes response to itself in OVX rodents receiving exogenous hormones, and it has generally been accepted that progesterone inhibits sexual behavior in guinea pigs under some circumstances (Blaustein and Wade, 1977a; Feder et al., 1968; Goy et al., 1966), its role in termination of sexual behavior during the estrous cycle (Hansen and Sodersten, 1978; Sodersten and Eneroth, 1981; Sodersten and Hansen, 1977) and pregnancy (Baum et al., 1979; Blaustein and Feder, 1979c; De Greef et al., 1981) of rats is unclear. Estradiol and progesterone are not the only sex hormones involved in the regulation of sexual behaviors. In fact, dihydrotestosterone is inhibitory to the induction of sexual receptivity (Baum et al., 1974; Blasberg et al., 1998; De Greef et al., 1981; Tobet and Baum, 1982), and it has been suggested as one of the factors that determines the duration of the period of sexual receptivity during the estrous cycle (Erskine, 1983). The time course of estradiol and progesterone action on sexual behavior may provide clues as to the underlying cellular processes by which hormones act in the brain. Although estradiol priming of response to progesterone generally takes about a day (Feder and Marrone, 1977; Green et al., 1970), an intravenous (IV) injection of progesterone in estradiol-primed rats facilitates the expression of lordosis within 1 h of injection (Glaser et al., 1983; Kubli-Garfias and Whalen, 1977; McGinnis et al., 1981; Meyerson, 1972). Therefore, progesterone exerts its neuronal influences on sexual behavior considerably more rapidly than estradiol, which suggests possible differences in the cellular mechanisms of each of the hormones. It is important to remember that, in addition to regulation by ovarian hormones, copulatory behavior is influenced by afferent input from mating. For example, the postural adjustments that accompany lordosis and facilitate intromission are made in

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the basic level of sexual receptivity. In addition, the lordosis rating, defined as the average intensity of each spinal dorsiflexion based on a four-point scale (Hardy and DeBold, 1972), is used to measure the magnitude of the lordosis response. In rats, the display of lordosis occurs briefly (approximately 0.5–1.5 s; Pfaff et al., 1973) beginning at the time of the mount, whereas in hamsters, the lordosis posture, once induced, is maintained for seconds-to-minutes (Carter and Schein, 1971) and in guinea pigs, it is held for 10–20 s (Goy and Young, 1957). During the estrous cycle, the sequential secretion of estradiol and progesterone results in a period of sexual behavior (heat; behavioral estrus) that is linked to the time of ovulation (Barfield and Lisk, 1974; Boling and Blandau, 1939; Collins et al., 1938; Powers, 1970). After the period of sexual behavior terminates, sexual receptivity is not expressed until the proestrous stage of the reproductive cycle returns with the next episode of secretion of estradiol followed by progesterone. Many experiments on the neuroendocrinology of feminine sexual behavior have been limited to assessing influences only on receptivity, so a bit of caution is warranted in generalizations to other aspects of sexual behavior. Removal of the ovaries causes rapid decline in circulating ovarian hormones, and consequently, cessation of the expression of feminine sexual behaviors (Boling and Blandau, 1939; Dempsey et al., 1936). Results of experiments, in which rats or guinea pigs were ovariectomized (OVX) at precise times during the estrous cycle, demonstrate that the hormonal requirements for the induction of an optimal level sexual behavior include a sufficient period of estradiol priming followed by exposure to progesterone ( Joslyn et al., 1971; Powers, 1970). Estradiol and progesterone are both necessary and sufficient for optimal levels of female sexual behaviors in OVX rats, guinea pigs, hamsters, and mice. Although each of these species (Carter et al., 1976; Crowley et al., 1978; Davidson et al., 1968; Mani et al., 1997) responds to treatment with estradiol alone under some conditions, it is sequential treatment with estradiol and progesterone that typically results in expression of feminine sexual behavior that most resembles that seen in estrous-cycling rodents (Beach, 1942; Boling and Blandau, 1939; Collins et al., 1938; Dempsey et al., 1936; Edwards, 1970; Tennent et al., 1980). Increasing doses of estradiol used for priming allow lower levels of progesterone to be

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Paracopulatory behaviors are species-typical behaviors, displayed by females, which arouse males and stimulate them to mount. These behaviors have variously been termed proceptive (Beach, 1976), precopulatory (Madlafousek and Hlinak, 1977), or solicitational (Erskine, 1989) behaviors. Paracopulatory behaviors exhibited spontaneously by estrous female rats during the normal course of mating, include hopping, darting, and ear wiggling (Beach, 1976; Madlafousek and Hlinak, 1977), a presenting posture (Emery and Moss, 1984a), a rapid sequence of approach toward, orientation to, and withdrawal from proximity to a sexually active male (McClintock and Adler, 1978), and production of ultrasonic vocalizations (White and Barfield, 1989). Blaustein and Erskine (2002) have used the term paracopulatory behaviors to obviate the assumptions about the female’s sexual motivation to initiate mating that are inherent in the other terms. All of these behaviors are expressed prior to and between mounts and occur repetitively during the mating session. The frequency and rate with which these behaviors are expressed are altered by ovarian steroids, by the rate at which a male copulates with a female, and by the specific experimental conditions under which mating is observed (Erskine, 1989). Paracopulatory behaviors are generally thought to be dependent on progesterone as well as estradiol. While lordosis is often observed within 1 h of progesterone treatment, maximum levels of paracopulatory behaviors are generally seen later, within about 2 h of treatment (Fadem et al., 1979; Glaser et al., 1983). Although estradiol alone induces paracopulatory (Fadem et al., 1979; Gorzalka and Moe, 1994) as well as copulatory (Boling and Blandau, 1939) behaviors in OVX rats, in most cases, progesterone of adrenal origin has been determined to be necessary for the paracopulatory behaviors (Gorzalka and Moe, 1994; Tennent et al., 1980). However, paracopulatory behaviors can be seen even in OVX-adrenalectomized rats administered high (Zemlan and Adler, 1977),

An additional component of female sexual behavior includes those behaviors which maximize the likelihood that pregnancy will occur, which we term progestative behaviors (Blaustein and Erskine, 2002). These behaviors, which occur throughout mating, regulate the frequency and timing of intromissions and ejaculations from males and include the female’s selection of males that are ready to ejaculate (McClintock et al., 1982a), the females’ postejaculatory interval which enhances sperm transport by preventing rapid displacement of the copulatory plug deposited by the male (McClintock et al., 1982b), and the female’s pacing of sexual stimulation, through intermittent approaches toward and withdrawals from the male (Bermant, 1961; Erskine, 1985; Gilman and Hitt 1978; Krieger et al., 1976; Peirce and Nuttal, 1961). They involve progressive short-term behavioral adjustments in the patterning of ongoing sexual contacts with males which ensure optimal reproductive success. The recognition that female rat sexual behavior contains elements through which females regulate the pattern of contact came from observations of feminine sexual behavior under seminatural (McClintock et al., 1982a; McClintock and Adler, 1978; McClintock and Anisko, 1982) and laboratory conditions (Bermant, 1961; Krieger et al., 1976; Peirce and Nuttall, 1961). The observation of progestative behaviors requires a test chamber or living environment in which females can avoid contacts with either the single male stimulus animal or an individual male in a multimale/multifemale social grouping. The most striking progestative behaviors in rats include those behaviors by which a female controls the timing of the intromissions that she receives from the male during copulation (Emery and Moss, 1984b; Erskine, 1985; Erskine et al., 1989; Gilman and Hitt, 1978; Pfaus et al., 1999). During a mating sequence, a female receives a number of mounts without intromission (mounts), mounts with penile intromission (intromissions), and several ejaculations from the male. In addition, female rats exhibit patterns of approach toward and withdrawal from males which occur in response to individual copulatory stimuli

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and in some cases, quite low doses of estradiol (SF Farrell and JD Blaustein, unpublished observations), suggesting that paracopulatory behaviors are not totally progesterone dependent.

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response to stimulation of the receptive field of the pudendal nerve (Adler et al., 1977; Komisaruk et al., 1972) during lordosis. Ovarian hormones influence the receptive field of the pudendal nerve that in turn influences the female’s response to mounting stimulation by the male.

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(mounts, intromissions, and ejaculations) and which serve to regulate, or pace, the types and amounts of vaginocervical stimulation (VCS) received during mating (Bermant, 1961; Erskine et al., 1989; Gilman et al., 1979; Krieger et al., 1976; McClintock et al., 1982a; Peirce and Nuttall, 1961). In a testing apparatus in which the male is restricted to one compartment, females actively pace their contacts with males by withdrawing to a neutral cage between mounts, intromissions, and ejaculations and then approaching the male for renewed copulation at predictable intervals (Bermant, 1961; Bermant et al., 1969; Bermant and Westbrook, 1966; Erskine, 1992; Frye and Erskine, 1990; Gilman et al., 1979; Krieger et al., 1976; McClintock et al., 1982a; Peirce and Nuttall, 1961). Females mated in small and/or undivided test arenas are unable to avoid contact with the male and thus receive nonpaced coital stimulation. Typically, paced mating occurs at a slower rate than nonpaced mating. The hallmark of pacing behavior is the differential pattern of approach and withdrawal, which occurs in response to changes in the intensity of sexual stimulation (Figure 1). In paced mating tests, females withdraw most frequently (percent exits) from the chamber containing the male after receiving an ejaculation than after receiving an intromission, and they withdraw least frequently after receiving a mount-without-intromission (mount). Females actively control the timing of copulatory stimuli by altering the rate at which they return to the male compartment after receiving each type of stimulation. Therefore, the rate at which they receive mounts, intromissions, and ejaculations from males is inversely proportional to the intensity of the specific mating stimulus (Erskine, 1985). The temporal patterning, or pacing, of VCS that females receive during mating is a critical determinant of whether the neuroendocrine responses required for pregnancy occur (Erskine et al., 1989; Erskine, 1995; Gilman et al., 1979), including secretion of sufficient progesterone from the corpus luteum to promote successful implantation. The ejaculatory stimulus has been demonstrated to be more effective than intromissions, or mounts-withoutintromission, in initiating the neuroendocrine changes required for pregnancy (O’Hanlon and Sachs, 1986). The imposition of a postejaculatory interval, when enforced by the female, is also progestative, insofar as this interval may ensure that maximum transport of sperm through the uterine cervix into the uterus has occurred (Adler and Zoloth, 1970).

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The role of estradiol and progesterone in pacing behavior has been studied. In general, nonreceptive rats do not pace (Brandling-Bennett et al., 1999). Although paracopulatory behaviors are dose dependent on progesterone, pacing may not be (Brandling-Bennett et al., 1999). However, it should be noted that in some situations, contact–return latencies were observed to be dose dependent on progesterone (Fadem et al., 1979; Gilman and Hitt, 1978). The reasons for discrepancy in the role of progesterone are unclear, but it may relate to the specific procedures used. In a direct comparison of paced mating during the estrous cycle and after 10 mg estradiol benzoate followed by 500 mg progesterone in OVX rats (Zipse et al., 2000), return latencies after mounts or

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Figure 1 Pacing of coital contacts by female rats in an escape–re-entry apparatus (top). The percentage of exits from and (bottom) the latency to return to the male compartment following mounts, intromissions, and ejaculations. *p < 0.001, significantly different from intromissions; {p < 0.001, significantly different from mounts. Reprinted from Erskine MS (1985) Effects of paced coital stimulation on estrous duration in intact cycling rats and ovariectomized and ovariectomizedadrenalectomized hormone-primed rats. Behavioral Neuroscience 99: 151–161, with permission from the American Psychological Society.

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2.2.4

Sexual Motivation

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While assessment of naturally occurring paracopulatory behaviors, such as darting, hopping, and earwiggling, goes beyond the recording of the lordosis response (Erskine, 1989), there is no evidence that these are indices of the female’s motivation to mate. Other tests, such as partner preference, the increasing barrier method, and conditioned place preference, have been developed to assess this more complex aspect of feminine sexual behavior (see Paredes and Vazquez (1999) for review). Level of sexual motivation can be assessed in partner preference tests in which a female is given an opportunity to seek proximity to a sexually active male over another stimulus animal (Clark et al., 1981a; Edwards and Pfeifle, 1983; Meyerson and Lindstrom, 1973). In general, rats in the proestrous/ estrous stage of the estrous cycle, or rats injected with estradiol, testosterone, or estradiol þ progesterone, are more likely to approach sexually active males (Clark et al., 2004; De Jonge et al., 1986; Edwards and Pfeifle, 1983; Meerts and Clark, 2006; Meyerson and Lindstrom, 1973), suggesting that these hormones increase the female’s sexual motivation. The female’s motivation is influenced by the ability of the male to copulate with her. Females show a stronger preference for males that are not allowed to copulate with them than with males that are (Clark et al., 2004). When given a choice between a castrated and an intact male, females spend more time with castrated males than with intact males (Broekman et al., 1988; Meyerson and Lindstrom, 1973). However, females exposed to males in a situation which restricts their sexual interactions (for instance, by keeping the males in a wire cage or by using a vaginal mask to prevent intromissions), spend more time with sexually active than with castrated, inactive males. These results suggest that females initially seek out contact with sexually active males, but that the mating stimulation they receive results in subsequent avoidance of this stimulation. This avoidance has also been observed in mating situations in which intensive mounting accompanied by intromission and ejaculation increases rejection behaviors and reduces the probability and intensity of lordosis (Hardy and Debold, 1971). Sexual motivation has also been assessed by the increasing barrier method in which an electric grid is positioned between the starting cage and the goal cage containing a stimulus male (McDonald and Meyerson, 1973). In general, female rats in the

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intromissions, but not ejaculations, were considerably longer in OVX, hormone-replaced rats than in ovaryintact rats. Although this issue of timing of rate of copulation is of tremendous importance, at this time, there is very little known about the cellular mechanisms which underlie these acute, transient changes in the female’s behavior. It should be noted, however, that estradiol and progesterone replacement cannot be given in a pattern that truly mimics the pattern of secretion during the estrous cycle. Mating stimulation by a male is reinforcing only when the females are allowed to pace the sexual interaction (Gilman and Westbrook, 1978), which is consistent with the idea that paced mating is a rewarding aspect of sexual behavior (Martinez and Paredes, 2001; Paredes and Vazquez, 1999). However, there are many inconsistencies in published reports of experiments, which have assessed motivation through the use of partner preference tests (Oldenburger et al., 1992). In elaborating the inconsistencies, Clark et al. (2004) emphasized the important influences that allowing or prohibiting contact with the male have on results. They argue for the importance of having a contact and no-contact condition when assessing sexual motivation using partner preference tests. Not all effects of steroid hormones on feminine sexual behavior are due to effects of the hormones in the brain; peripheral factors must also be considered, as was first discussed in Section 2.2.1 with regard to the postural adjustments that females make in response to the male’s stimulation of the receptive field of the pudendal nerve. For example, the systemic administration of the estrogen antagonist, ICI-182,780, which does not seem to cross the blood–brain barrier (Wade et al., 1993), was injected in rats treated with estradiol þ progesterone. While the antagonist was without effect on the expression of lordosis, it caused a lengthening of the return latencies after intromission and ejaculations in a paced-mating situation. Although the mechanism by which this occurs is not known, it has been suggested that peripheral, nociceptive changes are involved (Clark et al., 2003; Gardener and Clark, 2001). One explanation is that the anti-estrogen increased the aversive component of the genitosensory stimulation. This is consistent with the earlier findings of estradiol-induced increases in the sensory field of the pudendal nerve (Komisaruk et al., 1972) as well as the report that transection of this nerve disrupts pacing behavior in estradiol-treated rats (Erskine, 1992).

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mounts by a male. However, when a seminatural environment was used (Garey et al., 2002), mounts were often followed by darting to another part of the arena followed by return, perhaps similar to pacing in rats. Another report (Tomihara and Makino, 1991) suggests that lordosis is more likely when female mice approach males than when males approach females, which suggests that approach by females is a paracopulatory behavior.

2.3 Influence of Mating Stimulation on Long-Term and Short-Term Changes in Sexual Behavior and Neurons

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Many other nonhormonal factors associated with mating result in both short-term and long-term modulation of sexual behavior. For example, the intensity of copulatory and paracopulatory behaviors and the timing of progestative behaviors are modulated by feedback loops, which are activated in response to the sensory stimulation that the females receive from males during mating. Such stimulation includes mounts, intromissions, and ejaculations, as well as olfactory and auditory inputs.

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proestrous–estrous stage of the estrous cycle and OVX rats injected with estradiol are more willing to perform a higher number of grid crossings when approaching a sexually active male (Meyerson and Lindstrom, 1973), consistent with the idea that ovarian hormones influence the female’s motivation for sexual behavior. Conditioned place preference has been used to assess the female’s sexual motivation (Oldenburger et al., 1992). For example, it has been used to establish that estradiol- or estradiol þ progesterone-treated female rats prefer a compartment, which has previously been associated with sexual interaction. Consistent with some partner preference tests, these females prefer a compartment associated with a male who is not allowed to copulate. Also similar to what was seen in partner preference experiments, females develop a conditioned place preference for compartments in which they have been allowed to pace the mating, but not places where rate of copulation has been determined by the male (Gans and Erskine, 2003; Jenkins and Becker, 2003; Martinez and Paredes, 2001). Furthermore, estradiol alone is not sufficient to induce a conditioned place preference; progesterone is necessary (Gonzalez-Flores et al., 2004a; Paredes and Alonso, 1997; Paredes and Vazquez, 1999). Some of this work may have to be reevaluated in light of the recent report (Meerts and Clark, 2007) that nonpaced mating induces a conditioned place preference when a single male provides all of the intromissions, but not if multiple males are used to provide the full complement of intromissions.

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The hormonal sequence required for induction of sexual behavior in mice is similar to that required for guinea pigs, rats, and hamsters. However, the other species tend to respond to appropriate hormonal treatment at first exposure after ovariectomy, while mice generally do not (Mani et al., 1996, 1997; Rissman et al., 1997; Thompson and Edwards, 1971). In fact, mice typically require numerous exposures to estradiol and progesterone before they express high levels of sexual receptivity, and this varies by strain (Thompson and Edwards, 1971) and housing conditions (Laroche et al., 2005). Interestingly, weekly hormone treatments are not effective, unless they are accompanied by the experience of behavioral testing (Thompson and Edwards, 1971). Most research on mice assesses sexual behavior by way of the expression of lordosis in response to

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Vaginocervical Stimulation

VCS is a reproductively relevant stimulus that is an important component of mating stimulation received by females. Besides its influences on behavior, VCS influences luteinizing hormone (LH) release (Moss et al., 1977) and the twice daily surges of prolactin that then result in an extended period of diestrus (Gunnet and Freeman, 1983), called pseudopregnancy (Erskine et al., 2004) or the progestational state. Although not directly related to this chapter, the fact that VCS is a sufficient stimulus for formation of a maternal bond in postpartum ewes is evidence of the importance of VCS as a physiological stimulus (Kendrick et al., 1991b). Experimenter-administered VCS in rats can partially mimic the effects of intromissions by a male rat on reproductive physiology and behavior. This finding is interesting, because the probes that are used are typically smooth glass or plastic, and they provide pressure directly to the uterine cervix with mild distension pressure on the vaginal wall. In some cases, electrical stimulation of the cervix has been used (Gorospe and Freeman, 1981). In contrast, the rat penis is covered with keratinous spines

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Mating stimulation exerts both positive and negative feedback regulation of feminine sexual behavior. A positive effect is seen in the short-term enhancement of lordosis with intromissive stimulation. While female rats that are sexually receptive respond to appropriate stimulation of the flanks and perineum with the expression of the lordosis response, the presence of VCS by penile intromission results in a greater intensity of lordosis (Diakow, 1975). Similarly VCS, administered by a glass probe in OVX rats with or without subthreshold doses of estradiol, increases the likelihood of acute expression of lordosis in response to manual palpation of the flanks and perineum (Komisaruk and Diakow, 1973). Negative feedback effects are seen when the mating stimulation leads to subsequent decreases in sexual responsiveness. For example, during paced mating, withdrawal from the male occurs more

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2.3.2 Short-Term Effects of Mating Stimulation

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often, and the latencies of the female’s return to the male’s cage are longer when the antecedent stimulus is an intromission or an ejaculation than when it is a mount (Bermant, 1961; Bermant and Westbrook, 1966; Erskine, 1985). Thus, mounts, which include VCS, result in short-term inhibition of sexual responsiveness; that is, intromissions and ejaculations result in longer inter-intromission intervals than do mounts without intromission. Unfortunately, virtually nothing is known about the cellular mechanisms which underlie these rapid and transient changes. Over the course of a mating test in estrous-cycling female rats in estrus, the latency to return to the male’s cage increased as a function of the numbers of prior intromissions (Figure 2; Coopersmith et al., 1996a). The inter-intromission interval increases at a significantly greater rate during paced mating tests in which the female can control the timing of contact with males than in nonpaced tests. Therefore, when allowed to pace sexual contacts with males, there is a progressive lengthening in the rate at which female rats receive VCS, demonstrating that females modulate their own behavior in response to the prior stimulation received. The neuroanatomical areas involved in pacing in response to VCS have been studied. Lesions in the medial preoptic area, but not the medial amygdala or bed nucleus of stria terminalis (BNST), lengthened contact–return latencies only after intromissions and

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(Sachs et al., 1984; Taylor et al., 1983), which may heavily stimulate the vaginal wall, but may not actually contact the cervix directly during an intromission. Although experimenter-administered VCS has many of the same neuroendocrine effects as intromissions, it is unclear if it does so by the identical anatomical route.

9

20 Intromission number

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Figure 2 Cumulative time in seconds between intromissions during a 30-intromission mating test. P–P females received both the first 15 and the second 15 intromissions under paced mating conditions, and NP–NP females received the first 15 and the second 15 intromissions under nonpaced mating conditions. For NP–P females, the first 15 intromissions were nonpaced and the second 15 intromissions were paced. Reprinted from Coopersmith CB, Candurra C, and Erskine MS (1996a) Effects of paced mating and intromissive stimulation on feminine sexual behavior and estrus termination in the cycling rat. Journal of Comparative Psychology 110: 176–186, with permission from the American Psychological Society.

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2.3.3 Longer-Latency and LongerDuration Enhancement of Sexual Responding by Mating Stimuli

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Longer-latency and longer-duration influences of intromissive stimulation are also seen. An example of enhancement that is of longer duration is the acute facilitation of the lordosis response by experimenterapplied VCS or VCS with flank and perineal stimulation in rats that had received estradiol treatment or not that persists for several hours (Rodriguez-Sierra et al., 1975). When OVX rats are exposed intermittently to sexually active males, the level of their sexual receptivity increases over the first few hours (Auger et al., 1997; Foreman and Moss, 1977; Rajendren et al., 1990; Rajendren and Moss, 1993). In most earlier reports of mating-enhancement of sexual receptivity, rats that had been OVX, but not adrenalectomized, were used (Clemens et al., 1969; Hardy and Debold, 1973; Rajendren et al., 1990). Because adrenalectomy usually prevented this increase (Larsson et al. (1974), but cf. Zemlan and Adler (1977)), mere handling can sometimes substitute for mating (Hardy and Debold, 1973). Much of the earlier work showing enhancement of sexual responding as a consequence of nonintromissive mating and even handling was dismissed as being due to nonspecific influences on the adrenal gland, which secretes progesterone (Feder and Ruf, 1969). Although enhancement by repeated testing could be blocked by removal of the adrenals in some situations (Larsson et al., 1974), that is not always the case. In subsequent experiments, mating stimulation enhanced sexual behavior even in animals in which the adrenal gland was removed (Auger et al., 1997). The fact that enhancement of sexual behavior by mating stimulation occurs even in rats without

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ovaries and adrenals suggests that adrenal involvement was not an adequate explanation for these results. Study of the relative contribution of VCS as compared to flank and perineal stimulation provided by males suggested that the mating-enhancement is dependent in part upon intromissive stimulation (Bennett et al., 2001). Elimination of VCS (by placing tape over the vagina) blocked most of the enhancement; however, experimenter-applied VCS had only a minor influence on subsequent responding (Bennett et al., 2001). Nevertheless, on the basis of that work and work demonstrating the importance of intromissive stimuli/VCS on other aspects of reproduction and reproductive behavior, follow-up studies suggest that VCS may not be the proximate cause of mating enhancement in the repetitive mating situation. Mounts without intromissions were as effective in enhancing copulatory behavior as were mounts with intromission (Farrell et al., 2005; Ghavami et al., 2005). Furthermore, this enhancement is not limited to lordosis; dramatic increases in paracopulatory behaviors are observed as well. While both groups expressed high levels of paracopulatory behaviors (ear-wiggling, darting, and hopping), those receiving intromissive stimuli predictably expressed higher levels of rejection. These results have been replicated, so it is clear that mounting without intromission is a sufficient stimulus for enhancement of mating in estradioltreated, OVX–adrenalectomized rats in the absence of progesterone. This makes logical sense; enhancement precedes the receipt of intromissions because intromissions require that females display lordosis in response to mounts. Of course, they do not do this until they are receptive. There are clearly various routes to acute increases in the likelihood of lordosis or longer-latency enhancement. Experimenter-applied VCS with flank stimulation in the presence or absence of estradiol treatment (Rodriguez-Sierra et al., 1975) or in the absence of the pituitary gland (Rodriguez-Sierra et al., 1977) induces an acute lordosis response. Experimenter-applied VCS also induces a longer-duration enhancement of the likelihood of lordosis response to manual flank and perineal stimulation. Estradiolprimed, OVX–adrenalectomized rats exposed to sexually vigorous males do not require intromissive stimulation for enhancement of sexual behavior. Therefore, although VCS can induce lordosis, even in the absence of ovarian or hypophyseal hormones, mating stimulation from male rats does not require intromissive stimulation.

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ejaculations (Guarraci et al., 2004; Yang and Clemens, 2000). Although other areas are likely to be involved, the medial preoptic is an essential neuroanatomical site in the processing of sensory information relating specifically to VCS that results in changes in paced-mating behavior. It should be noted that knife-cuts parasagittal to the ventromedial nucleus of the hypothalamus (VMN) that inhibit estradiol þ progesterone-applied lordosis in response to a male, are without effect on cervical-probing-applied lordosis (Pfeifle et al., 1980), potentially suggesting a different neural pathway for lordosis that is stimulated in this manner.

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In another form of negative feedback, the receipt of intromissions and ejaculations, but not mounts alone, results in abbreviation of the period of estrus in rats (Boling and Blandau, 1939; Erskine, 1985; Lodder and Zeilmaker, 1976; Pfaus et al., 2000; Reading and Blaustein, 1984), guinea pigs (Goldfoot and Goy, 1970; Roy et al., 1993), and hamsters (Carter, 1972, 1973; Carter et al., 1976; Carter and Schein, 1971; Ramos and Debold, 1999). Allowing females to pace sexual contacts increases the inhibitory effect of intromissive stimulation on estrous duration in estrouscycling rats (Coopersmith et al., 1996a; Erskine, 1985; Erskine et al., 1989). In many reports, the period of sexual receptivity has been defined as the time during which a female responds to appropriate mating stimulation with the lordosis response. However, in a testing situation which allows females to passively avoid males and/ or demonstrate rejection behaviors (presumably the situation in nature), heat termination is preceded by the presence of active and passive rejection behaviors. For example, in a bilevel testing chamber in which females can escape from the male (Pfaus et al., 2000), experimenter-applied VCS induces both passive (remaining away from the male) and active rejection behavior toward the male by 16–20 h, prior to the loss of the lordosis response. Although van der Schoot et al. (1992) concluded that rats do not show heat abbreviation as a consequence of intromissive stimulation, the latter result suggests that those investigators may not have waited a sufficiently long period of time to test for abbreviation. Mating-induced heat abbreviation in rats is mediated by the pelvic nerve, because pelvic neurectomy blocks it (Lodder and Zeilmaker, 1976). However, in guinea pigs, transection of the pelvic, pudendal, and genitofemoral nerves is without effect (Slimp, 1977). This has been taken as evidence for a non-neural route for the information from the genitals to the brain in guinea pigs. However, because the vagus nerve also plays a role in conveying sensory information from

2.4 Hormonal Regulation of Steroid Hormone Receptors and Feminine Sexual Behavior

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In order to discuss the integration of afferent, environmental information with hormonal signals, it is first necessary to briefly review a bit of what is known about the role of steroid hormone receptors and their regulation in the expression of feminine sexual behavior. Furthermore, an understanding of the hormonal regulation of receptors and involvement of receptors in behavioral response may provide clues regarding mechanisms by which the environment may influence them. The history of the development of this field is quite interesting, and the reader is referred to Blaustein and Erskine (2002) for a more in-depth discussion. A great deal has been learned recently about the various forms of each receptor and molecular biology and genetics of the receptors, and the reader is referred to other chapters in this volume.

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2.3.4 Longer-Latency Inhibition of Sexual Behavior by Mating Stimuli: Heat Abbreviation

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the vagina/cervix to the brain (Cueva-Rolon et al., 1996; Guevara-Guzman et al., 2001; Komisaruk et al., 1996), the possibility that this route is involved in guinea pigs cannot be excluded. However, Roy et al. (1993) then showed that, while an ejaculation by an intact male abbreviated the period of sexual receptivity, an ejaculation by a gonadectomized male with hormone replacement did not. Although this study suggests that in guinea pigs, unlike rats and hamsters, heat abbreviation may be dependent upon a factor in intact males’ ejaculate, it also cannot be excluded that the nature of the mechanical stimulus delivered by the intact male was quantitatively different from that of the gonadectomized male. Furthermore, this latter study is in conflict with the earlier study (Goldfoot and Goy, 1970) in which it was reported that either a male or experimenter-applied VCS resulted in heat abbreviation.

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There are numerous influences, besides hormones, on the moment-to-moment levels of sexual responding. While these factors have not been as well studied as the response to hormones, they may have dramatic influences on sexual behavior and ultimately, fertilization, so they should receive equal attention.

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Estrogen Receptors

Although there is not total consistency among all of the studies that have been performed on regulation of estrogen receptor (ER) a and ERa mRNA levels by estradiol, generally estradiol downregulates this receptor in most neuroanatomical areas (DonCarlos et al., 1995; Greco et al., 2001; Lauber et al., 1991; Meredith et al., 1994; Shughrue et al., 1992; Simerly and Young, 1991). Most studies find that estradiol also downregulates the second ER discovered, ERb, in some neuroanatomical areas and is without effect

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receptivity confirming the critical role of ERa in copulatory behavior in mice (Kudwa and Rissman, 2003). Interestingly, although there is no evidence of an important role of ERb in sexual behavior in adults, a role has been suggested in defeminization of the brain and behavior (Kudwa et al., 2005). Further support for involvement of ERa in feminine sexual behavior comes from the recent study using RNAi silencing of ERs in the VMN (Musatov et al., 2007). The presence of receptors in a tissue has often been looked at as having a permissive or all-or-none influence on response. However, in some experiments, an attempt has been made to relate receptor levels more appropriately to sensitivity to the hormone. Yet another level of regulation of hormone sensitivity lies in steroid receptor coregulators. Although a great deal of work has been done on the role of coregulators in modulation of transcriptional activity of ERs in vitro, few experiments have examined them within the context of ERs and behavior. Work using antisense oligonucleotides mRNA for steroid receptor coactivators (SRCs) in rats suggests that SRC-1 and cAMP response element-binding protein (CBP) act together to modulate the induction of sexual receptivity by estradiol (Molenda et al., 2002), as well as progesterone (Molenda-Figueira et al., 2006). Likewise, others (Apostolakis et al., 2002) have suggested the importance of SRC-1 and SRC-2 in the cellular action of estradiol in the induction of feminine sexual behavior in rats and mice. Although in vitro studies suggest that the relative expression levels of the coactivators and corepressors determine cell-specific, appropriate, and graded responses to steroid hormones (Lonard and O’Malley, 2005), there has, to date, been no work on this subject in the brain and on behavior. The pattern of hormonal exposure is a critical variable in determining response to hormones. Rats do not have to be exposed to estradiol continuously during the priming period in order to express sexual behavior. Two small pulses of a very low dose of estradiol are more effective in inducing feminine sexual behavior in response to progesterone than a single large injection or continuous exposure (Clark and Roy, 1983; Parsons et al., 1982; Sodersten et al., 1981; Wilcox et al., 1984). The behavioral effects of each pulse can be blocked by a protein synthesis inhibitor (Parsons and McEwen, 1981) or pentobarbital anesthesia (Roy et al., 1985). The particular proteins modulated in response to each injection may differ ( Jones et al., 1986). This topic will be discussed again in Section 2.4.3.

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in others (Greco et al., 2001; Osterlund et al., 1998; Patisaul et al., 1999; Suzuki and Handa, 2004). The inconsistencies in the literature with respect to the regulation of ERs by estradiol are to be expected, because a wide variety of techniques have been used, each with their own strengths and weaknesses. There are also numerous, important methodological differences, such as doses of estradiol used, duration of exposure to hormone, time since ovariectomy, etc. Furthermore, there is heterogeneity in the regulation of each form of ER, not just among neuroanatomical areas, but among different neurons in a particular area. Perhaps most importantly, ERa and ERb are not regulated in parallel in all tissues (Greco et al., 2001). Long-term deprivation of steroid hormones by ovariectomy decreases the response of rats and guinea pigs to induction of sexual behavior by estradiol and progesterone (Beach and Orndoff, 1974; Clark et al., 1981b; Czaja et al., 1985; Delville and Blaustein, 1989). Long-term ovariectomy (Clark et al., 1981b; Delville and Blaustein, 1989; Parsons et al., 1979) results in a decrease in the concentration of estradiol-induced, hypothalamic progestin receptors (PRs) in rats. Although estradiol-binding studies have typically shown no change in brain ERs after long-term ovariectomy (Clark et al., 1981b; Parsons et al., 1979), both long-term (Liposits et al., 1990) and short-term (Shughrue et al., 1992) ovariectomy results in an increase in ERa immunoreactivity and/or mRNA. The necessity of ERs for the actions of estradiol on sexual receptivity has been confirmed in three ways: estrogen antagonists, which block the binding of estradiol to ERs, ER gene-disrupted mice (ER knockouts; ERKO’s), in which the gene for ERs has been disrupted, and most recently RNAi silencing of ERs in specific brain regions (Musatov et al., 2006). The results of each approach are consistent with the conclusion that ERs are essential for the effects of estradiol on the expression of sexual receptivity. KO strains of mice have been developed in which the gene for each (Krege et al., 1998; Lubahn et al., 1993) or both (Couse and Korach, 1999) ERs is disrupted. Targeted disruption of the ERa gene completely eliminates hormonal induction of feminine sexual behavior (Ogawa et al., 1998; Rissman et al., 1997). However, disruption of the ERb gene is without apparent effect in OVX, hormone-injected mice (Kudwa and Rissman, 2003), but it extends the period of behavioral estrus and enhances receptivity in estrouscycling mice (Ogawa et al., 1999). Double KO mice (abERKOs) with disruption of both ERs are not only infertile, but also show decreased levels of sexual

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As with ERs, the reader is referred to the review by Blaustein and Erskine (2002) in the first edition of this volume for an extensive discussion of the history of studies of PRs in the brain.

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2.4.2.1 PRs and facilitation of feminine sexual behavior

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The characterization of neural PRs in the early 1980s led us to propose that PRs are essential in the facilitation of sexual behavior by progesterone. This hypothesis predicted that sensitivity to progesterone is determined by the concentration of unoccupied PRs available in neurons involved in progesteronefacilitated sexual behavior, and response is dependent on an adequate concentration of activated PRs in those cells. An increased concentration of PRs (e.g., after estradiol priming) should increase the sensitivity of the neural substrate for progesterone, presumably by increasing the concentration of receptors that become activated in response to progesterone treatment. Likewise, a decreased concentration of unoccupied PRs result in decreased sensitivity to progesterone. After treatment of guinea pigs (Blaustein and Feder, 1979b) or rats (Moguilewsky and Raynaud, 1979; Parsons et al., 1980) with estradiol, the concentration of PRs in the hypothalamus increases, as does responsiveness to progesterone. The increased concentration of receptors and behavioral responsiveness to progesterone are both transient (Blaustein and Feder, 1979b; Clark et al., 1982; Parsons et al., 1980). Similarly in OVX animals, the concentration of unoccupied PRs in the hypothalamus increases during proestrus in response to estradiol (McGinnis et al., 1981). The timing of the duration of the period of sexual receptivity is referable to the regulation of activated PRs in particular neurons (Blaustein and Olster,

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1989). The period of sexual receptivity for each species is rather tightly regulated, with heat in guinea pigs lasting about 8 h (Young, 1969), and heat in rats lasting approximately 14 h (Blandau et al., 1941). Injection of a behaviorally effective dose of progesterone in estrogen-primed guinea pigs and rats (Blaustein and Feder, 1980; McGinnis et al., 1981; Rainbow et al., 1982) or the preovulatory secretion of progesterone during the estrous cycle of rats (Rainbow et al., 1982) causes the rapid binding to PRs, including those in the hypothalamus and preoptic area. The presence of activated PRs in the hypothalamus–preoptic area after progesterone injection correlates well with the expression of sexual behavior (Blaustein and Feder, 1980). This temporal concordance suggested that the expression of lordosis requires maintenance of elevated levels of occupied PRs, and that termination of sexual behavior is due to loss of these receptors. Physiological manipulations which increase the duration that hypothalamic PRs remain occupied also extend the duration of the period of sexual behavior (Blaustein, 1982a; Brown and Blaustein, 1985; Joslyn and Feder, 1971). A variety of techniques have been used to demonstrate that PRs are necessary for the facilitation of sexual behavior by progesterone – injection of progestin antagonists, antisense oligonucleotides to PR mRNA, and PR KO strains of mice. Each will be discussed briefly; more extensive discussion can be found in the first edition of this volume (Blaustein and Erskine, 2002). Systemic injection (Brown and Blaustein, 1984; Richmond and Clemens, 1986) or intracranial application (IC; Etgen and Barfield, 1986; Mani et al., 1994a) of the progestin antagonist, RU 486, inhibits the facilitation of sexual behavior by progesterone in rats and guinea pigs. The fact that a very high dose of progesterone, but not cortisol, facilitates the expression of sexual behavior in guinea pigs treated with RU 486 (Brown and Blaustein, 1984) demonstrated that the inhibition was not due to RU 486’s well-known ability to block glucocorticoid receptors (GRs) (Moguilewsky and Philibert, 1984), and it suggested that the inhibition by RU 486 was due to specific blockade of PRs (Brown and Blaustein, 1986). In one case, the progesterone antagonist, RU 486, was found to have progesterone-like effects on feminine sexual behavior (Pleim et al., 1990). This is not unexpected, because antagonists are seldom pure, and RU 486 has progestin agonist-like effects in some situations (Vegeto et al., 1992) and ERb antagonist effects in others (Sathya et al., 2002; Zou et al., 1999).

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It should be noted that not all ER-mediated responses require the prototypical estrogen response elements on specific genes. A gene knockin mouse model in which a mutant ERa that does not bind to estrogen response elements, Glidewell-Kenney et al. (2007) showed negative, but not positive, feedback to estradiol, suggesting that negative feedback does not require binding of ERa to an estrogen response element. While expression of masculine sexual behavior in males was shown to require binding to estrogen response elements (McDevitt et al., 2007), feminine sexual behavior has not been studied.

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2.4.2.2 Downregulation of PRs leads to estrous termination and the refractory period

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Which cellular events lead to termination of hormone-applied sexual behavior? During the estrous cycle, as in OVX animals administered hormones exogenously, there is a temporal correlation between decreased blood levels of progesterone and termination of behavioral estrus (Blaustein and Feder, 1980; Feder et al., 1968). However, the two events are not causally related. Extended maintenance of elevated blood levels of progesterone by supplemental progesterone treatment in guinea pigs prolongs heat duration slightly (Blaustein and Brown, 1985; Morin and Feder, 1973), but it terminates despite maintenance of elevated progesterone levels. Progesterone downregulates its own receptors. Loss of behavioral response can typically be attributed to either a declining concentration of activated/ unoccupied hypothalamic PRs or the absence of a sufficient level of progesterone to interact with the particular level of unoccupied receptors. The decline

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in concentration of unoccupied PRs can come about in a variety of ways. A decrease in estradiol levels results in the loss of induction of PRs. In addition, exposure to progesterone downregulates PRs. Both processes typically occur in tandem. The expression of sexual behavior requires the maintenance of activated PRs. Injection of the progestin antagonist, RU 486, during the period of sexual behavior, shortens the duration of behavioral estrus in guinea pigs (Brown and Blaustein, 1986) and rats (Brown et al., 1987). Abbreviation of heat duration is believed to be secondary to the antagonist-applied loss of activated hypothalamic PRs (Brown et al., 1987; Brown and Blaustein, 1986). It must be emphasized here that, with the cell fractionation techniques used in these studies, unactivated receptors were found in the cytosol fractions of homogenates, while activated receptors localized to cell nuclei. For the purpose of discussion, we are simplifying and referring to occupied, cell nuclear receptors as activated and receptors found in the cytosol as unactivated. The refractory period, which follows termination of sexual receptivity (Blaustein and Wade, 1977b; Goy et al., 1966; Nadler, 1970), may be a result of the same mechanisms as cause heat termination – downregulation of PRs by progesterone. First, during the refractory period, the concentration of hypothalamic PRs is depressed in relevant brain areas (Blaustein and Feder, 1979a; Blaustein and Turcotte, 1990; Moguilewsky and Raynaud, 1979; Parsons et al., 1981), and progesterone treatment results in low levels of activated PRs (Blaustein, 1982b; Blaustein and Feder, 1980). Second, supplemental estradiol offsets the progesterone-applied refractory period, such that animals regain responsiveness to the second progesterone injection (Blaustein and Wade, 1977b; Joslyn and Feder, 1971; Nadler, 1970; Shivers et al., 1980). In guinea pigs, the supplemental estradiol injection also offsets the decrease in the concentration of unoccupied PRs, resulting in high levels of occupied PRs in response to progesterone (Blaustein, 1982a). Third, the refractory period can be overcome by a large dose of progesterone (Blaustein, 1982b; Hansen and Sodersten, 1979), despite the presence of low PR levels. This high dose causes a large increase in progesterone-occupied, hypothalamic PRs, while a lower, behaviorally ineffective, dose does not (Blaustein, 1982b). Therefore, under a variety of conditions, there is a strong relationship between the level of activated, hypothalamic PRs, and the expression of lordosis.

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Because most antagonists are not totally specific, other techniques have been used to test the necessity of PRs for progesterone function in sexual behavior. Infusion of antisense oligonucleotides to PR mRNA, which inhibits PR synthesis, into the cerebral ventricles (Mani et al., 1994c) or ventromedial hypothalamus (VMH; Ogawa et al., 1994; Pollio et al., 1993) blocks facilitation of copulatory behavior and paracopulatory behavior by progesterone. Similarly transgenic strains of mice with a targeted disruption of the PR gene (PRKOs; Lydon et al., 1995) are completely unresponsive to progesterone for facilitation of sexual behavior (Mani et al., 1996). There are two isoforms of PR, a long form (PR-B) and a truncated, short form (PR-A), both synthesized from the same PR gene with alternate transcription and translation initiation sites (Conneely et al., 1989; Kastner et al., 1990). Until recently, it was not possible to study the role of each isoform. However, as will be discussed in Section 2.5.3, PR-A has an essential role in progesterone-facilitated sexual behavior. Although the emphasis of this chapter is on the mechanisms by which afferent input influences the genomic mechanisms involved in hormonal regulation of sexual behavior, as will be discussed in other chapters, nongenomic influences of progestins play a role in regulation of feminine sexual behaviors as well (Debold and Frye, 1994).

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Although the role of membrane receptors in the hormone action in the brain is of potential importance, it is beyond the scope of this chapter, and is discussed in detail in other chapters in this volume and in recent reviews (Ronnekleiv and Kelly, 2005). Their role in feminine sexual behavior will only be discussed briefly here. Little is known about the cellular basis for the enhanced response to pulsed exposure to estradiol discussed in Section 2.4.1. However, estradiol conjugated to bovine serum albumin (BSA), which makes the compound impermeable to cell membranes, can substitute for either pulse of estradiol-17b (Kow et al., 2005), suggesting a role for membrane ERs in priming by estradiol. The actions of the estradiol– BSA conjugate can be mimicked by compounds that activate either protein kinase A (PKA) or PKC, suggesting involvement of these two intracellular signaling pathways in the action of estradiol on feminine sexual behavior, as has been shown for the action of estradiol on neuronal electrophysiology (Kelly and Wagner, 1999). Interestingly, anesthesia during either pulse of estradiol in this type of procedure blocks the induction of feminine sexual behavior (Roy et al., 1985), suggesting that neural activity is required for either pulse to be effective. It should be noted that experiments using protein conjugates of steroid hormones must be interpreted very cautiously because of the possibility that the protein could be cleaved from the steroid molecule (Stevis et al., 1999; Vasudevan and Pfaff, 2008) and because the position of the protein on the steroid can have tremendous effects on function (Temple and Wray, 2005). Membrane ERs have been implicated in the regulation of feminine sexual behavior by other experiments. For example, a biotinylated form of estradiol interacts with metabotropic glutamate receptors in the medial preoptic nucleus, resulting in the expression of feminine sexual behavior in rats (Dewing

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progesterone is a consequence of this downregulation. Fewer PRs are available to bind progesterone, so animals tend to be unresponsive. Thus, the refractory period can be seen as a logical extension of the cellular processes that lead to heat termination, and regulation of PR levels and/or activation provides mechanisms by which neurotransmitters or the environment may regulate sexual behavior and other neural responses

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Downregulation of neural PRs and ERs, as with other steroid receptors (Nawaz et al., 1999), seems to be due to activity of the 26S proteasome (CamachoArroyo et al., 2002; Villamar-Cruz et al., 2006). Inhibition of 26S proteasome activity not only stabilizes the concentration of PRs in the hypothalamus and preoptic area, but it also prevents the progesterone-applied refractory period in female rats (Gonzalez-Flores et al., 2004b). These results provide a possible cellular mechanism for the downregulation of PRs, which leads to behavioral refractoriness to progesterone. Just as there had been question about the presence of a progesterone-applied refractory period in rats (Zucker, 1967), there have been conflicting reports of progesterone involvement in termination of behavioral estrous in rats. Although rats may not become completely insensitive to progesterone, they do, in fact, become hyposensitive to progesterone after termination of behavioral estrus during the estrous cycle as well as in the OVX rats treated with estradiol and progesterone (Sodersten and Hansen, 1979). The idea that heat termination and the refractory period are due to loss of activated PRs may explain the conflicting opinions concerning progesterone’s role in estrous termination (Barfield and Lisk, 1974; Hansen and Sodersten, 1978; Powers and Moreines, 1976). Perhaps the critical variable is the concentration of PRs, not progesterone itself. Inhibition of protein synthesis in the medial preoptic area, in hamsters (Ramos and Debold, 1999), or systemically, in guinea pigs (Goldfoot and Goy, 1970), blocks mating-induced abbreviation of the period of sexual receptivity. Likewise, inhibition of protein synthesis delays heat termination (Blaustein et al., 1982). We have suggested that heat termination is referable to decreases in the concentration of activated PRs in relevant neurons. None of these studies assessed the effects of inhibition of protein synthesis on activated PR levels. Another cellular event that may be related to estrous termination is the downregulation of ERs by progesterone under some conditions (Attardi, 1981; Bethea et al., 1996; Blaustein and Brown, 1984; Brown and MacLusky, 1994; Smanik et al., 1983). A decrease in the concentration of ERs would be expected to decrease the synthesis rate of PRs, which would then further contribute to a low level of PRs and decreased response to progesterone. To summarize, the evidence suggests that termination of the period of sexual receptivity after progesterone treatment results from downregulation of PRs, and the ensuing period of hyposensitivity to

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information and steroid hormone-sensitive systems have focused on the regulation of these receptors. The finding that catecholaminergic activity influences the concentrations of neural sex steroid receptors in rat and guinea pig brain (Blaustein, 1992b) suggested that environmental stimuli might regulate the concentration of steroid receptors in neurons involved in feminine sexual behavior, and consequently, behavioral response to hormones. Drugs which either inhibit norepinephrine (NE) synthesis (dopamine-b-hydroxylase inhibitors) or which block noradrenergic receptors (e.g., aadrenergic antagonists) typically decrease the concentration of ERs in some neural areas (Blaustein et al., 1986; Blaustein, 1987; Blaustein and Letcher, 1987; Blaustein and Turcotte, 1987a; cf. Malik et al., 1993; Montemayor et al., 1990) and/or inhibit the induction of unoccupied, hypothalamic PRs by estradiol (Clark et al., 1985; Nock et al., 1981; Thornton et al., 1986), and a-adrenergic agonists reverse this suppression. Lesioning noradrenergic inputs to the hypothalamus tends to decrease ER concentrations in the hypothalamus (Montemayor et al., 1990). Finally, under some conditions, stimulation of dopamine (DA) receptors increases the concentration of ERs in the brain (Blaustein and Turcotte, 1987b; Gietzen et al., 1983; Thompson et al., 1983; Woolley et al., 1982). In some cases, these treatments have been shown to decrease feminine sexual behavior (Montemayor et al., 1990; Nock and Feder, 1984). An anatomical substrate for integration between catecholaminergic neurons and steroid hormoneresponsive neurons has been described. Some ER-containing neurons receive apparent input from catecholaminergic neurons (Heritage et al., 1977, 1980), and tyrosine hydroxylase-ir and dopamine-b-hydroxylase-ir (DBH-ir) varicosities are sometimes found closely associated with PR-ir or ER-ir neurons in the preoptic area and hypothalamus (Blaustein and Turcotte, 1989; Brown et al., 1990), including the ventrolateral hypothalamus of female guinea pigs (Tetel and Blaustein, 1991). The fact that ER-ir cells with closely associated DBH-ir varicosities stain more darkly for ERs than ER-ir neurons lacking this association suggests noradrenergic regulation of levels of ERs in a population of cells in this area (Tetel and Blaustein, 1991). Other neurotransmitters have been reported to regulate steroid receptors. For example, muscarinic agonists and antagonists regulate the levels of neural ERs (Lauber, 1988a,b; Lauber and Whalen, 1988), and NE (Maccari et al., 1992) and serotonin (Mitchell

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et al., 2007); ERa interacts with mGluR1a in the arcuate nucleus resulting in the internalization of m-opioid receptors in the medial preoptic and consequently, the expression of feminine sexual behavior. With the exception of the recent work of Kow et al. (2005), mechanistic studies have typically focused either on the role of nuclear ERs and PRs acting as transcriptional regulators or on the role of membrane receptors for the steroid receptors in regulation of sexual behavior. However in vitro, the ERa and the ERb gene each direct the synthesis of receptors that may become associated with membranes, and are capable of signaling through the mitogen-activated protein (MAP) kinase pathway (Razandi, 1999; Levin, 2005; Wade et al., 2001). This finding raises an important question about the studies that have either used hormone antagonists, antisense oligonucleotides, or targeted gene disruption to test the involvement of steroid receptors acting as transcription factors. If the same ER and PR genes that direct synthesis of the receptors that act as transcription factors also direct synthesis of membrane receptors in the brain, then the conclusions of some of these experiments could be open to reinterpretation. That is, these manipulations could disrupt membrane receptors as well. ERa (Blaustein, 1992a; Blaustein et al., 1992; Milner et al., 2001; Wagner et al., 1998) and PRs (Blaustein et al., 1988; Clarke et al., 2000; Watson and Gametchu, 1999) have each been observed in extranuclear locations within the hypothalamus, including distal dendrites and axon terminals (Blaustein et al., 1992). In some cases, they have been observed associated with synaptic densities and plasma membranes (Blaustein et al., 1992; Blaustein, 1994; Clarke et al., 2000), perhaps consistent with the idea that cell nuclear receptors can be directed to membrane sites.

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2.5 Crosstalk between Neurotransmitters and Steroid Hormone Receptors

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2.5.1 Neurotransmitters Influence Concentrations of ERs and PRs

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Because of the critical (albeit not exclusive) role of steroid hormone receptors in the mechanisms of action of steroid hormones on feminine sexual behavior, studies of integration between afferent

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In addition to regulation of concentration of steroid hormone receptors, steroid hormone receptors can be activated by a variety of intracellular signaling pathways in the absence of steroid hormones (Blaustein, 2004; Cenni and Picard, 1999). After first showing that the chick ovalbumin upstream promoter (COUP) receptor could be activated in vitro by DA (Power et al., 1991a), Power et al. (1991b) then showed that DA agonists can also activate PRs. Ligand-independent activation of steroid receptors provides a potential mechanism by which afferent input from the environment might activate steroid receptors in particular populations of neurons. Mani et al. (1994a) first reported that dopaminergic agonists can activate PRs in neurons in vivo in the absence of progesterone. Intracerebroventricular (ICV) administration of D1-specific, DA agonists substituted for progesterone for facilitation of sexual behavior in estradiol-primed rats (Figure 3; Mani et al., 1994a). Facilitation by dopaminergic agonists was blocked by progesterone antagonists, antisense

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Ligand-Independent Activation of

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2.5.2 PRs

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et al., 1992; Seckl and Fink, 1991) each influence the concentrations of GRs. Some work has shown that specific afferent connections influence steroid receptor levels; anterior roof deafferentation knife-cuts increase the concentration of ERs in the mediobasal hypothalamus and increase the behavioral response to estradiol, while decreasing the concentration in the septum (Chen et al., 1992). Conversely, the concentration of ERs in the medial amygdala in female rats increases after olfactory bulb removal (McGinnis et al., 1985). Cues from the social environment can in some cases regulate steroid hormone receptors and hormonal response. Exposure of female prairie voles to the odors of males induces estrous behavior (Carter and Getz, 1985) and increases the concentration of ERs in the preoptic area (Cohen-Parsons and Roy, 1989). Similarly, social conflict ( Johren et al., 1994), perinatal handling (O’Donnell et al., 1994), and other stressors (Meaney et al., 1996) each influence the concentration of GRs. Although studies of the regulation of ovarian steroid receptors lag behind, a great deal of progress has been made in a related field – the cellular processes underlying the influences of maternal licking and grooming on hippocampal GRs. Handling causes changes in serotonin release (Meaney et al., 1994). Activation of 5-HT7 receptors (LaPlante et al., 2002) in turn activates NGFI-A expression, which then regulates GR expression via regulation of the GR promoter (Szyf et al., 2005). In this case, maternal care results in DNA methylation of the GR promoter, which then causes long-term changes in receptor expression. In a case with more relevance to feminine sexual behavior, differences in maternal care also alter ERa expression in the medial preoptic area (Champagne et al., 2003) via changes in methylation of the ERa-1b promoter (Champagne et al., 2006). The altered regulation of ERa has been linked to subsequent changes in maternal behavior; however, potential influences on feminine sexual behavior have not been studied. Although the cellular processes by which environmental influences modulate steroid receptor levels are just beginning to be elucidated, it is clear that the principle of localized regulation of steroid hormone receptor concentrations by neurotransmitters is a common means of integration of environmental input with steroid signaling pathways. This regulation may occur by interaction of particular neurotransmitters with their receptors on steroid receptor-containing neurons.

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Figure 3 Effect of ICV-administered dopamine D1 agonist, SKF 38 393 on lordosis response in PR knockout (PRKO) mice. Ovariectomized PRKOs and their wild-type littermates were primed weekly with estradiol and progesterone and tested weekly. On week 6, they were injected with estradiol benzoate followed by ICV infusion of SKF 38 393 (50 ng) or saline vehicle control or estradiol benzoate followed by saline or progesterone 48 h later. Statistically significant differences were seen in SKF- and progestin-facilitated responses of the PRKOs compared to the wild type (*P