Sexual Differentiation of Brain & Physiology:

Pfeiffer (1933)

• Known: The pituitary and ovary were important for ovulation.

• Question: Are there differences between males and females in ability to support ovulation?

• Concluded: Males and female rats differ in their ability to support ovulation.

• Concluded (although incorrectly): Male rat’s pituitary was unable to support ovulation.

ovarian fragments(able to ovulate)

eyesof female

rats

showovulation

eyesof male

rats

could notshow

ovulation

Sexual Differentiation of Brain & Physiology:

Pfeiffer (1933)

• Question: Are testicular secretions during first few days of life

involved in making male rats unable to support ovulation?

• Concluded: Testicular secretions act early in development to make male rats (and even female rats) unable to support ovulation.

newborn male castratedat birth

male couldshow

ovulation

as adult,w/own ovariesor implanted

ovaries

femalecould not

showovulation

implantedw/ovariesas adults

newbornfemale

implantedw/testicular

tissueat birth

Sexual Differentiation of Brain & Physiology:Harris (1952)

• Known:

– species that are “induced ovulators”--ovulation is triggered by copulation (e.g.,

rabbit, cat); implied that the NS was involved in the process of ovulation (via

sensory input associated with copulation)

– hypothalamus (in the brain) was the likely source of control

• Question: Are differences in the ability to support ovulation between male

and female rats the result of differences in the pituitary or brain?

• Concluded: The difference between male and female rats in the ability to

support ovulation was not due to differences in the pituitary.

• Instead, differences must lie within the brain--hypothalamus.

female rat with amale pituitary

could showovulation

removedpituitary

from female rat

implantedpituitary

frommale rat

Ovulation and GnRH Surge in Rats:

Ovulation:

• as follicles develop in ovary

– increasing levels of estrogen

are released

– in female rats, increases in

estrogen lead to a GnRH

surge (positive feedback)

– GnRH surge leads to LH

surge

– LH surge leads to ovulation

• male rats are unable to show

a GnRH surge in response to

increases in estrogen

GnRH Neuron

HYPO

ANTPIT

OVARY

FSHLH

Estrogen

GnRH+

GnRH: gonadotropin-releasing hormoneFSH: follicle stimulating hormoneLH: luteinizing hormone

Sexual Differentiation of Brain & Behavior:

Males & females of a variety of species show differences in behavior.

• One example is the display of lordosis by female rodents during mating.

• Lordosis--posture shown by female rodents in which the female arches her

back to elevate the rump and head.

• Background:

– estrogen and progesterone have activational effects on female sex behavior

– ovariectomize (OVX) adult female rats-->females don’t show lordosis

– OVX adult female rats + [estrogen followed by progesterone]-->female will

show lordosis

– castrate adult male rats + [estrogen followed by progesterone]-->don’t see

lordosis

• Basic observation: the nervous system of adult males and females are

different in their behavioral responsiveness to hormones.

Sexual Differentiation of Brain & Behavior:Phoenix and associates (1959)

• Studied female sex behavior in guinea pigs--display of lordosis.

• Question: Does exposure to androgens early in development alter the

display of female sex behavior in the adult?

• Concluded: Testicular secretions act early in development to make male rats (and androgenized females) unable to show female sex behavior (lordosis). Instead, androgen exposure “organizes” the brain so that males will show male sex behavior in the adult.

newborn male

castratedat birth

male canshow lordosisin response to

mounting

givenestrogen &

progesteroneas an adult

female cannotshow lordosisin response to

mounting

givenestrogen &

progesteroneas an adult

newbornfemale

givenandrogens

at birth

Process of Sexual Differentiation:

Role of androgens in males:• permanently masculinize the brain

– male specific responses (sex behavior)

• permanently defeminize the brain– inability to support ovulation (no GnRH surge in male rats)– inability to show female sex behavior in presence of ovarian hormones

What about females?• Dogma: no androgens-->brain & behavior becomes feminized (passive)• However, recent evidence suggests that some estrogen is needed for

development of female brain and display of female-specific responses.– block interaction of estrogen with ER (antagonist) during development-->

individual that shows no female sex behavior nor ovulation

MALES

feminized

demasculinized

FEMALES

masculinized

defeminized

“active”

Critical Periods:

• brief “windows of time” when steroids can alter development of body and nervous system

• multiple “windows” are seen during development

– prenatal, perinatal and postnatal periods

• these “windows” reflect transient changes in steroid levels and require the presence of steroids receptors (ARs or ERs) and possibly converting enzymes (aromatase or 5α-reductase)

Rats:

• injections of testosterone on day of birth or up to around the 10th day of life can render a female anovulatory and unlikely to display lordosis

• however, injections of testosterone by day 12th (or after) has little effect on these measures

Paradox:

Early exposure of newborn females to elevated levels of estrogen could

masculinize the brain, behavior & physiology of these individuals as adults.

Estrogens are largely formed by the ovary and ovaries are found in females.

• Why would an ovarian hormone cause masculinization of the brain and behavior?

• Aromatization is important for masculinizing the brain in some species (rats).

– testosterone estrogen estrogen-ERs produces an effect

– need testosterone, aromatase (which will produce estrogen), and ERs

Testosterone(precursor)

estrogen

aromatase

• If estrogens can masculinize the brain, how do females normally escape

masculinization?

– ovaries of female fetuses (in utero) secrete very little steroid

– both male and female fetuses see high levels of estrogen (source: maternal ovaries,

adrenal glands)

– normally, the brains of male fetuses are masculinized/defeminized but the brains of

females are not--why?

– presence of a protein: -fetoprotein (AFP)

– AFP binds to estrogen and appears to block estrogen’s ability to reach the brain

– however, AFP does not bind to testosterone; therefore, testosterone can enter the

brain and be converted to estrogen via aromatase--masculinization of brain

– the ability of exogenous estrogen to masculinize the brain requires--high levels--

which presumably swamps the buffering capacity of AFP allowing some estrogen

to reach the brain

– estrogen and sexual differentiation:

• no estrogen ”unisex brain”

• some estrogen feminized brain

• testosterone or lots of estrogen masculinized brain

Species Differences:

There are species differences in what hormones masculinize and defeminize the

brain and behavior.

• In guinea pigs and primates:– testosterone or other androgens (dihydrotestosterone) must interact with ARs to

masculinize/defeminize the brain

– of interest, a homologue to AFP has been identified in primates but does not bind to estrogen (in rats, the AFP does bind to estrogen)

rats/hamsters

male sexbehavior

guinea pigs/primates

dependenton

aromatization

not dependenton

aromatization

Species Differences:

There are species differences in what processes are masculinized and

defeminized.

male rats

Masculinized:

male primates

male sexbehavior

true

Defeminized: no positivefeedback response

to estrogen; nosupport of ovulation

not true

Sexual Dimorphisms within Adult Nervous System:

Sexual dimorphisms have been observed in the following parameters:

• number of neurons

• size of neurons (large or small)

• number and shape of synapses

• length and branching of dendrites

• amount and type of neurotransmitters, enzymes and receptors that are expressed

Examples (shown in class):

• SDN-POA (sexually-dimorphic nucleus of the preoptic area)--sex differences in the size of nucleus

• SNB (spinal nucleus of the bulbocaveronosus)--sex differences in the presence/absence of brain nucleus

How do hormones affect sexual differentiation?

• drive neuronal cell differentiation (number of cells born), cell migration

and/or cell survival

– Ex. SDN-POA

• promote outgrowth of dendrites and axons of specific neurons

• provide target-derived neurotrophic action

– Ex. SNB

• regulate the expression of specific molecules--neurotransmitters, enzymes

and receptors

Hormones--Cell Survival:Ex. Sexually Dimorphic Nucleus of the Preoptic Area (SDN-POA) (in rats)

• SDN-POA is 3-5 times larger in males than in females

• aromatization of testosterone to estrogen is important for masculinization

• originally thought--androgens were important in stimulating the number of

neurons born in males that will migrate to, and form, the SDN-POA (book)

• however--more recent data suggest that exposure to androgens perinatally act

to increase the number of neurons that survive in males than in females

• how can we follow cell birth/survival? tritiated thymidine autoradiography

• current view: (following injection of 3H-thymidine on day 18 of gestation)

– PN4--# neurons in SDN-POA: males=androgenized females= females

– PN7--# neurons in SDN-POA: [males=androgenized females]>females

– neurons are lost in females at PN7 and PN10; exposure to testosterone (from E20

to PN10) can prevent this loss

– males have larger SDN-POA because more neurons survive into adulthood, and

also because of an increase in volume not associated with addition of more

neurons--increase in cell size (larger) and/or more connections

Tritiated Thymidine Autoradiography:• neuroblasts in the ventricular

zone will divide, differentiate into neurons, and migrate to specific areas in the brain

• during cell division, DNA is being synthesized

• 3H-thymidine will be incorporated into newly synthesized DNA

• if 3H-thymidine is injected on day 18 of gestation, then all neurons “born” on day 18 will have radioactive DNA with cell’s nucleus

• can identify “birthdate” of neurons by exposing brain sections to X-ray film or by dipping sections in a photographic emulsion

day 17

VentricularZone

day 18 day 19

DevelopingBrain

[3H]-thymidine

Gestation

• radioactivity will expose the photographic emulsion

• can learn: how many neurons are born on a given day, where they migrate, and if they survive into adulthood

• Ex. Spinal Nucleus of the Bulbocavernosus (SNB) (in rats)

• SNB is present in males and absent in females

• SNB neurons are motoneurons that innervate muscles attached to the penis (perineal muscles)

• early in development: androgens increase survival of muscles which leads to survival of motoneurons innervating the muscles

• later in development: androgens act subsequently to increase size of neurons (larger); ARs are expressed in SNB motoneurons at later time than ARs expressed in muscle

• administration of estrogen cannot masculinize SNB motoneurons (aromatization is not important); thus, androgens act directly at ARs to masculinize SNB system

SNB

Perinealmuscles

Hormones--Target-Derived Neurotrophic Function:

duringcritical period

of development,muscles can bind

androgens while themotoneurons

cannot

secrete a“retrograde factor”

that leads to survival

of neurons

Example Question:

What structures within the nervous system would be masculinized or feminized

in a male rat with testicular feminization mutation?

SDN-POA

normalmale

SNB

masculinized(large)

male withTFM

masculinized(large)

Sexual Differentiation of the Human Nervous System:Sexually dimorphic nuclei have been described within preoptic area of humans:

• INAH-1 (intermediate nucleus of the anterior hypothalamus--cell group #1)– nucleus is larger in males than in females

– sex difference develops postnatally (not present at birth)

– after 4 years of age, the number of neurons in nucleus die in females, but remain the same in males; androgens are believed important for survival of these neurons

– function is not currently known

• INAH-3 (interstitial nucleus of the anterior hypothalamus--cell group #3)– nucleus is larger in males than in females

– not clear how hormones affect its development

– nucleus is also larger in heterosexual men than homosexual men; suggested that this nucleus might be important for sexual orientation

– sexual orientation can be viewed as a sexually dimorphic response: masculine preference is for female partners, and feminine preference is for male partners

– smaller INAH-3 in homosexual men=feminine preference for male partners

– elevated androgens in utero may act to masculinize sexual orientation

– evidence that 37% women with CAH rate themselves as bisexual or homosexual while only 7% women without the disorder rate themselves similarly

– cautionary note: sex behavior in humans can be affected by many factors

Sexual Differentiation of the Human Nervous System:Onuf’s nucleus is also sexually dimorphic in humans:

• Onuf’s nucleus is the homologue to the rat SNB

• motoneurons within Onuf’s nucleus innervate roughly the same group of

muscles within the perineum: the bulbocavernosus (BC) and ischiocavernosus

(IC) muscles; humans (and other higher mammals) lack levator ani muscle

• men have larger BC and IC muscles and more neurons within Onuf’s nucleus

than females

• subtle effect: sex difference between men and women is smaller than the

difference reported between male and female rats; in female rats, these

muscles are lost and so are the motoneurons

Sexual Differentiation of the Human Nervous System:Sex differences have also been reported in cognitive function:

• men are lateralized in auditory function: most men can hear better with their

right ear than with their left

• in contrast, women tend to be less lateralized in auditory processing, hearing

equally well with right and left ears

Women exposed to a synthetic estrogen in utero show higher levels of

lateralization in auditory function:

• in 1950s and 1960s, diethylstilbestrol (DES--synthetic estrogen) was given to

pregnant women to prevent miscarriages

• women exposed to DES in utero were more lateralized in word detection than

their sisters that were not exposed to the drug

• in females, exposure to exogenous estrogen can masculinize auditory function

• in males, testosterone is most likely aromatized to estrogen which leads to

lateralization of auditory function