Chp 4 Hormonal Influences on Male Sex Behavior

Chp 4: Hormonal Influences on Male Sex Behavior Male sex behavior can be divided into two main components: • precopulatory behavior: – production of ultrasonic vocalizations – chemoinvestigation (sniffing of the female’s anogenital region) • copulatory behavior: – mounts: male mounts the female from behind – intromissions: male, with an erected penis, inserts penis into the female’s vagina – ejaculations: explusion of a copulatory plug--composite of different secretions from prostate gland, seminal vesicle and coagulating gland plus sperm; this composite will “coagulate” forming a plug that serves to keep the sperm within female’s reproductive system and increase likelihood of fertilization

Sequence of Precopulatory and Copulatory Events: copulation chemoinvestigation mount intromission ejaculation (repeated X times) postejaculatory interval (PEI) satiety postejaculatory interval (PEI): refractory period following ejaculation in which male does not engage in sex behavior (seconds to minutes) satiety: refractory period following a series of ejaculations in which a male will not engage further in sex behavior with a given female

Quantification of Precopulatory & Copulatory Behaviors: How to analyze male sex behavior? • amount of time engaged in sniffing odors (chemoinvestigation) • latency to mount; number of mounts • latency to intromit; number of intromissions; – interintromission interval--length of time between intromissions • latency to ejaculate; number of ejaculations • postejaculatory interval: length of time from an ejaculation to the next intromission

Sexual Motivation In addition to precopulatory and copulatory behaviors, we we can also consider the male’s desire to engage in sex behavior. • Sexual motivation: male’s inclination to seek out and approach a female for the purpose of mating. Tests of sexual motivation: • latency to mount: – mount quickly-->really motivated – mount slowly-->not very motivated – has problems--? ? • Train a male to press a bar to gain access to a female--how many bar presses will a male make? (more bar presses=greater motivation) • Separate males and females across an electrified grid--how much electrical current will a male withstand to gain access to a female? (greater current-greater motivation)

Hormonal Influences on Male Sex Behavior • In females, transient elevations of estrogen and/or progesterone during the estrous cycle play an important role in activating female sex behavior. • In males, testosterone (and/or its metabolites) also play an important role in activating male sex behavior. – Testosterone secretion increases at puberty: development of secondary sex characteristics, increased muscle mass, production of sperm, and development of an interest in sexual activity. – However, testosterone levels do not show similar cyclical changes in levels reminiscent of the female estrus cycle.

Hormonal Influences on Male Sex Behavior Testosterone is the major androgen secreted by the testes. • aromatization of testosterone to estrogen: – important for activating male sex behavior in the rat (and in many other species, although not all) – involve aromatase and ERs • reduction of testosterone to dihydrotestosterone: – important for penile erections and intromissions in the rat – involve 5 -reductase and ARs • testosterone as testosterone: : – important for aspects of sexual motivation – involve ARs

Testosterone Secretion in Males: • Gn. RH Neuron - HYPO ANT PIT • In interstitial space (testis): – LH acts at the Leydig cells to secrete the testosterone (“steroidogenesis”) – LH-->increase levels of c. AMP FSH LH TESTES – Gn. RH stimulates release of LH and FSH from anterior pituitary + Gn. RH neurons secrete Gn. RH into the median eminence: • Inhibin Testosterone Gn. RH: gonadotropin-releasing hormone FSH: follicle stimulating hormone LH: luteinizing hormone In seminiferous tubules: – at puberty, FSH and testosterone act at Sertoli cells to initiate the production of sperm (“spermatogenesis”) – FSH -->increase levels of c. AMP – FSH also stimulates the production of inhibin from Sertoli cells which inhibits release of FSH from anterior pituitary

Hormonal Influences on Male Sex Behavior Testosterone is the major androgen secreted by the testes. • aromatization of testosterone to estrogen: – important for activating male sex behavior in the rat (and in many other species, although not all) – involve aromatase and ERs • reduction of testosterone to dihydrotestosterone: – important for penile erections and intromissions in the rat – involve 5 -reductase and ARs • testosterone as testosterone: : – important for aspects of sexual motivation – involve ARs

Species Differences: Male Sex Behavior • In rats (and hamsters): – estrogen is critical for activating copulation--mounts, intromissions and ejaculations ) – dihydrotestosterone is critical for penile reflexes • In some species (lizards, mice &guinea pigs): – dihydrotestosterone can stimulate male sex behavior • In ferrets: – testosterone plays selective role in sexual motivation • In primates: – testosterone may also have a selective effect on sex behavior not mediated by estrogen or dihydrotestosterone

Peripheral Mechanisms: Androgens are critical for penile erections and deposition of the copulatory plug: • androgens stimulate the size and number of sensory receptors on the surface of the glans penis • dihydrotestosterone (DHT) activates penile erections when rats are restrained on their backs • penile erections and deposition of the copulatory plug are controlled (in part) by motoneurons within spinal nucleus of the bulbocavernosus: – SNB innervates penile musculature – DHT acts to increase survival of penile musculature followed by survival of SNB motoneurons (during development) – in the adult, DHT also affects the morphology of SNB neurons--increasing size of cell bodies and increasing length of dendrities – Note: penile erections and ejaculation are also controlled by the autonomic nervous system.

Central Mechanisms: In the male rat: • estrogen acts to stimulate copulation--mounts, intromissions and ejaculations – aromatization is important for this process! • MPOA plays an important role the effects of estrogen: – sexually dimorphic area of the preoptic area (SDA-POA): sexual learning – dorsal MPOA: critical for stimulation of mounts, intromissions and ejaculations – dopaminergic inputs--activating mounts, intromissions and ejaculations • amygdala plays an important role in facilitating sex behavior--the amygdala attributes “salience” to cues associated with sex behavior – corticomedial amygdala--pheromones to activate behavior – basolateral amygdala--learned responses (e. g. , bar pressing to gain access to female) • dopaminergic inputs into the nucleus accumbens--sexual motivation • dopaminergic inputs into the striatum--general locomotion (e. g. , chasing female)

MPOA In the rat: • Aromatization of testosterone to estrogen is important for sexual differentiation of the brain and activation of male sex behavior. • The MPOA is an integrative center for hormonal stimulation of male copulation. – Large lesions of the MPOA eliminate mounting, intromissions and ejaculations. – Electrical stimulation of the MPOA stimulates these responses. – Castrate an adult male rat and he will stop engaging in copulation. – If you administer testosterone to a castrated male, he will again engage in copulation. – If you administer testosterone or estrogen within the MPOA (with DHT to act peripherally), normal male copulation will be seen-->MPOA is critical site!! • The MPOA sends projections to neurons within the midbrain and brainstem, which subsequently project to neurons in the spinal cord; 2 effects take place: – activation of autonomic centers that control process of erection and ejaculation – activation of motoneurons that control mounting and pelvic thrusts

MPOA Anatomical Subdivisions: • Study by Heimer and Larsson (1966/67): – Large bilateral lesions of the MPOA eliminated mating. • Study by Arendash & Gorski (1983): – Is the SDN-POA the critical brain site for control of male copulation? – No: in sexually experienced males lesions of the SDN-POA had no significant effect on male copulation. – Instead, bilateral lesions of the dorsal MPOA produced substantial, long-term decreases in number of mounts, intromissions, and ejaculations compared to sham-lesioned control males. • Study by De Jonge et al. (1989): – Revisited role of SDN-POA in male sex behavior--found an effect! – In sexually naïve males, bilateral lesions of the SDN-POA resulted in an increased latency to first mount, intromission and ejaculation (took longer to initiate copulation) – However, this decrement in sexual performance disappeared after repeated testing.

Amygdala: Corticomedial Amygdala (CMA): • CMA: plays an important role in linking pheromones to activation of male sex behavior – CMA receives olfactory stimulation from the vomeronasal organ and olfactory mucosa (sensory structures responsd to odors; project to the olfactory bulbs). – Pheromones are secreted by female that can stimulate chemoinvestigation and copulation. – CMA projects to the MPOA. – Lesions of the vomeronasal organ and olfactory mucosa or bilateral lesions of the CMA can eliminate male sex behavior--effect is similar to bilateral lesions of MPOA. Olfactory Bulbs CMA MPOA brainstem spinal cord “Copulatory reflexes”

Amygdala: Basolateral Amygdala (BLa): • BLa: implicated in associative learning that feeds into motivation circuits. • You can train a rat to bar press to gain access to an estrus female. – If you lesion the BLa, the male rat will not show bar pressing for the female. – However, if you place the receptive female in the chamber with the male, he readily copulates with her. – It is thought that the association between bar pressing to gain access to a female rat (for copulation) has been lost. • The BLa, via connections within the nucleus accumbens, is thought to play a role in sexual motivation--searching out a female (e. g. , bar pressing) in order to mate.

Nucleus Accumbens The nucleus accumbens (NAcc) has been linked to motivational processes. • Mogensen (1980) suggested that the NAcc serves as the “limbic-motor interface” – a system concerned with translating emotional (“salient”) stimuli into motor output Limbic System: Prefrontal Cortex BLa “associative learning” NAcc Ventral Pallidum MPOA Motor System: “Motor Responses” a) foraging for a female b) bar pressing for a female Basal ganglia Mesencephalic Locomotor Region

Central Mechanisms: In the male rat: • estrogen acts to stimulate copulation--mounts, intromissions and ejaculations – aromatization is important for this process! • MPOA plays an important role the effects of estrogen: – sexually dimorphic area of the preoptic area (SDA-POA): sexual learning – dorsal MPOA: critical for stimulation of mounts, intromissions and ejaculations – dopaminergic inputs--activating mounts, intromissions and ejaculations • amygdala plays an important role in facilitating sex behavior--the amygdala attributes “salience” to cues associated with sex behavior – corticomedial amygdala--pheromones to activate behavior – basolateral amygdala--learned responses (e. g. , bar pressing to gain access to female) • dopaminergic inputs into the nucleus accumbens--sexual motivation • dopaminergic inputs into the striatum--general locomotion (e. g. , chasing female)

Dopaminergic Cell Groups: Dopamine Cell Bodies General Effect incertohypothalamic DA cell group MPOA ventral tegmental area (VTA) nucleus accumbens substantia nigra mounts, intromissions and ejaculations (copulatory reflexes) display of motivated behavior dorsal striatum sensorimotor coordination (locomotion)

Dopamine The levels of dopamine increase within these areas during mating: • Study by Pfaus et al. (1990): – in vivo microdialysis was used to follow levels of dopamine (and its metabolites) within nucleus accumbens and dorsal striatum during copulation • in vivo microdialysis involves measurement of molecules present in the extracelular space through diffusion across a semipermeable membrane at the base of a probe implanted into brain – nucleus accumbens: levels of dopamine increased significantly during placement of a sexually experienced male in a sex chamber (with odors of prior matings present), with additional increases observed with placement of a female behind a screen, and then when the male was allowed to mate with female – dorsal striatum: levels of dopamine increased significantly only during interaction between the male and female (copulation) – concluded: DA released in the nucleus accumbens (from VTA) is involved in motivation processes (prior to, and in preparation of, the actual act of copulation); DA released in the striatum is involved in actual physical act of copulation (sensorimotor coordination)

Dopamine The levels of dopamine increase within these areas during mating: • Studies by Hull et al. (1995, 1997): – in vivo microdialysis was used to follow levels of dopamine (and its metabolites) within MPOA during copulation – MPOA: levels of dopamine increased significantly during precopulatory period in which a female is placed behind a perforated barrier (male can see, hear and smell female but not mate with her), and then further increases in dopamine occur when the male can interact with the female – concluded: DA released within MPOA is involved in sexual motivation (DA is increased prior to, and in preparation of, the actual act of copulation), and copulatory responses (DA increases further during the actual act of copulation)

Central Mechanisms--Dopamine Data from lesions and dopamine release during behavior provide insight into functional roles of various brain regions to sex behavior: • dorsal striatum: – lesions of dopamine neurons in substantia nigra produce Parkinsonism symptoms: difficulty initiating movements, slowness of actions and tremor – DA levels increase significantly during the act of copulation – dorsal striatum is thought to play a role in sensorimotor coordination (pursuit of female) • MPOA: – lesions of MPOA block display of mounts, intromissions and ejaculations – administration of DA antagonists into the MPOA can inhibit display of copulation – DA levels increase prior to, and during, the act of copulating with a receptive female – MPOA is thought to play a role in both sexual motivation and copulation – MPOA receives pheromone-associated information from CMA; this information provides one source of excitatory information linked to both sexual motivation and performance

Central Mechanisms--Dopamine Nucleus Accumbens (NAcc): – lesions do not block the display of copulation – however, blocking input into the NAcc by bilateral lesioning the BLa can block bar pressing in order to gain access to a receptive female (discussed in terms of sexual motivation) – DA levels increase prior to, and during, the act of copulating with a receptive female – however, DA levels also increase in NAcc in response to feeding, drinking, drug selfadministration – Nacc is thought to play a role motivational processes and reward

Hormonal Regulation Testosterone, and/or its conversion to estrogen, is important not only for stimulating copulation but also for motivational processes. If you train a male to bar press to gain access to a female, you can study the effects of specific brain lesions and compare those effects to that produced by castration. Sexual performance: • bilateral lesions of the MPOA, but not BLa, eliminate copulation (low % of rats engaged in copulation) • castration also dramatically decreases % rats copulating Motivation processes: • bilateral lesions of the BLa, but not the MPOA, greatly reduce bar pressing to gain access to a receptive female • castration also dramatically decreases bar pressing

Hormones and Dopamine Regulation Does castration alter dopaminergic neurotransmission to effect changes in male sex behavior? Answer--yes, testosterone facilitates dopamine release! Studies by Alderson & Baum (1981); Mitchell & Stewart (1989): • castration decreases the level of dopamine within the nucleus accumbens • administration of testosterone, or estrogen plus dihydrotestosterone, increases dopamine levels within this nucleus Study by Hull et al. (1997): • castration decreases release of dopamine within the MPOA in response to a precopulatory period (female behind barrier) or interactions with an estrous female • some castrates (1 week) showed dopamine release in MPOA and engaged in sex behavior (short-term response; eventually all castrated males will stop mating) • administration of testosterone proprionate restored release of dopamine within MPOA in response to precopulatory period and facilitated mating

Opioid and Male Sex Behavior Opioids have been shown to inhibit male sex behavior. Study by Hughes & colleagues (1988): • infusion of B-endorphin into the medial preoptic area causes a reduction in male’s mating performance • infusion of naloxone, an opioid receptor antagonist, can facilitate sexual performance and block inhibitory effect of B-endorphin MPOA male copulation Arcuate Nucleus naloxone B-endorphin neurons

Opioid and Male Sex Behavior Opioids have been shown to stimulate male sex behavior. Mehrara and Baum (1990): • Design: – conditioned place preference is a test of motivation (desire to spend more time on one side of a chamber than the other) – established conditioned place preference in male rats given the opportunity to mate on only one side of the chamber – how much time would the males spend on the side of the chamber in which they had mated – several variables: 1) sham-operated vs. castration, 2) given naloxone at varying doses or saline, and 3) testing the animals 7 days after surgery or 14 days

Opioid and Male Sex Behavior Opioids have been shown to stimulate male sex behavior. Mehrara and Baum (1990): • Results: – administration of naloxone decreased time spent in the chamber associated with mating (e. g. , loss in conditioned place preference) – administration of naloxone also decreased time spent in the chamber associated with mating in males 7 days after castration, but not 14 days, after castration • Conclusion: – opioids act to facilitate conditioned place preference (blocking opioid activity can blockthis response) – the ability of opioids to facilitate conditioned place preference is lost at 14 days postcastration – opioids act to stimulate dopamine release from neurons within the VTA; dopamine neurons in VTA project to NAcc; NAcc is involved in motivational processes

Opioid and Male Sex Behavior Opioids have been shown to stimulate male sex behavior. opioids Nucleus Accumbens VTA + + Motivation (e. g. , conditioned place preference) dopamine neurons

Neurons & Behavior How to study role of specific groups of neurons in specific behavioral responses? • compare the behavior of lesioned animals to sham-operated controls – lesions can be made with electrical current (electrolytic) or with neurotoxins (neurochemical) • compare the behavior of different groups of animals following the administration of drugs that either increase or decrease the level of a specific neurotransmitter: – lesion specific neurons: 6 -hydroxydopamine is used to lesion dopamine neurons – use agonists to stimulate activity at specific receptors – use antagonists to block activity at specific receptors • follow the activation of groups of neurons at various endpoints of behavior: – release of neurotransmitters – expression of molecules that serve to “mark” activated neurons • expression of c-fos proto-oncogene

c-fos Expression c-fos expression is used as a marker of neuronal activation: • levels of c-fos m. RNA and Fos protein are low in control, unhandled animals • if you stimulate an animal--handling, mating, stress, etc. , you will see significant increases in the level of these molecules • the rise and fall occurs quickly: – c-fos m. RNA peaks by 30 -60 minutes, and declines to basal levels within 2 hours – Fos protein peaks by 1 -2 hour, s and declines to basal levels within 4 hours • the transient nature of c-fos expression as well as the low levels basally enables detection of these molecules to serve as a “marker” of neuronal activation • c-fos expression serves to couple “inputs” (neuronal or hormonal) with gene transcription: – Fos protein forms a dimer with Jun protein--called “AP-1” factor – AP-1 factor binds to specific DNA sequences (AP-1 site) to control transcription of genes

Components of Mating Kollack-Walker & Newman (1997): • Experimental Design: – sexually experienced male hamsters were exposed to female hamster vaginal secretion (FHVS) for 10 minutes, or allowed to mate to different endpoints of copulation: 5 intromissions, 1 ejaculation, 5 ejaculations, long intromissions – long-intromissions: characterized by a period of extended intravaginal thrusting during which no sperm transfer occurs; the occurrence of long intromissions is believed to reflect a state of sexual satiety (turning off male sex behavior) – following FHVS exposure or copulation, males were kept in the mating arena (up to one hour) until they were anesthetized, perfused with fixative, their brains removed from the skull and sectioned on a freezing microtome – a series of sections from each animal were processed for Fos immunocytochemistry – the number of Fos-immunoreactive neurons were determined for specific brain regions in each animal, averaged per group, and compared statistically

Components of Mating Kollack-Walker & Newman (1997) • Results: – significant patterns of activation were observed within medial amygdala (Me. A), bed nucleus of the stria terminalis (BNST) and medial preoptic area (MPOA) following: FHVS exposure alone, mating to 1 ejaculation, and mating to multiple ejaculations – in particular, neuronal cell “clusters” in the caudal part of the posterior medial nucleus of the amygdala (c. Me. PD) were seen in 67% of males mating to 5 ejaculations, and in 100% of males mating to long intromissions • Conclusion: – different patterns of c-fos expression were observed in males that engaged in different levels of sexual activity (chemoinvestigation, copulation) • Question: Does the selective pattern of neuronal activation seen following multiple ejaculations reflect a state of satiety or simply a phenomonon of experiencing multiple ejaculations?

Sexual Satiety Parfitt & Newman (1997) • Experiment #1: Do the number of ejaculations prior to the occurrence of long intromissions make a difference in the pattern of c-fos expression? • Design: – compare pattern of Fos-immunostaining within sexually experienced male hamsters mated to long intromissions in 2 groups: 1) rested male hamsters (rested 1 week), and 2) male hamsters mated to long intromissions on 4 consecutive days prior to experimental day – rested male hamsters show more ejaculations (on average 9 ejaculations) while repeatedly mated males show fewer ejaculations (on average 4 ejaculations) • Results: (focus on neuronal cell “clusters” in c. Me. PD) – males mated to long intromissions in both groups (rested male hamsters and repeatedly mated male hamsters) showed the presence of neuronal cell clusters in c. Me. PD • Conclusion: – the number of ejaculations does not alter presence of neuronal cell“clusters” in c. Me. PD

Sexual Satiety Parfitt & Newman (1997) • Experiment #2: Does mating to only 1 ejaculation produce different patterns of neuronal activation in males that differ in their proximity to sexual satiety? • Design: – compare pattern of Fos-immunostaining within sexually experienced male hamsters mated to only one ejaculation in 2 groups: 1) rested male hamsters (rested 1 week), and 2) male hamsters mated to long intromissions on 4 consecutive days prior to experimental day • Results: (focus on neuronal cell “clusters” in c. Me. PD) – following mating to only 1 ejaculation, repeatedly mated males showed the presence of neuronal cell “clusters” within c. Me. PD, while the rested males did not • Conclusion: – the number of ejaculations are not critical for the occurrence of neuronal cell “clusters” within c. Me. PD, rather the proximity to satiety was the critical factor – neuronal cell “clusters in c. Me. PD may reflect development of a state of satiety!

The Coolidge Effect: • males recently mated with a particular female appear uninterested in further copulation (sexual satiation) • however, if a new female is introduced, then the male will continue to mate • thus, the introduction of novel females can extend a male’s sexual motivation and performance

Sexual Satiety Summary: • different patterns of c-fos expression are observed in male hamsters mated to different endpoints of copulation--female odors, 1 ejaculation and multiple ejaculations • neuronal cell “clusters” in c. Me. PD: their appearance is dependent upon how close a male is to sexual satiety – clusters are present in all males mated to long intromissions (LI) – clusters are present after only 1 ejaculation in repeatedly mated males (4 ejaculations away from LI) but not in rested males (9 ejaculations away from LI) • thus, neuronal cell “clusters” in c. Me. PD may reflect development of a state of satiety • in the literature, there is evidence that lesioning this part of the medial amygdala in female hamsters can produce a significant increase in the duration of copulation [Takahashi & Gladstone (1988) Behav. Neurosci. , 102: 268 -275]

Seasonality in Reproduction • Seasonal breeders: engage in sexual activity during certain times of the year. • Reproductive axis also varies with the breeding season. During Breeding Season Out of Breeding Season Gn. RH LH & FSH testes testosterone Display of sex behavior No display of sex behavior • Seasonal breeders: hamsters, sheep, deer, zebra finches, ferrets • Nonseasonal breeders: rats, humans, primates • Similar events within the reproductive axis are observed in males and females of species that are seasonal breeders.

Seasonality in Reproduction • The breeding season for different species varies, and is dependent upon an interaction between environmental factors and length of gestation. • Environmental factors: food availability, temperature--signaled by changes in day length (photoperiod). • Goal: ensure that the offspring are born under conditions optimal for their survival. Ex. Sheep Ex. Ferrets Breeding season in fall Breeding season in spring Daylength is decreasing Daylength is increasing Gestation period: 5 months Gestation period: 41 days Newborn lambs will be born in spring Newborn ferrets will be born in spring

Seasonality in Reproduction • Seasonality can affect how the brain responds to gonadal steroids: house under short day photoperiod male hamsters castrate replace testosterone house under long day photoperiod • How well does administration of exogenous testosterone restore male sex behavior? • Testosterone is most effective in stimulating male sex behavior in males housed under long day photoperiod compared to males housed under short day photoperiod.