2. 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)
eyes
of female
rats
show
ovulation
of male
could not

3. 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.
implanted
w/ovaries
as adults
newborn male
castrated
at birth
male could
show
ovulation
implanted
w/testicular
tissue
at birth
as adult,
w/own ovaries
or implanted
ovaries
female
could not
show
ovulation
newborn
female

4. 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 a
male pituitary
could show
ovulation
removed
pituitary
from
female rat
implanted
male rat

5. Ovulation and GnRH Surge in Rats:
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 +
GnRH
ANT
PIT
FSH LH
OVARY
Estrogen
GnRH: gonadotropin-releasing hormone
FSH: follicle stimulating hormone
LH: luteinizing hormone

6. 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.

7. 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.
given
estrogen &
progesterone
as an adult
male can
show lordosis
in response to
mounting
newborn
male
castrated
at birth
given
estrogen &
progesterone
as an adult
given
androgens
at birth
female cannot
show lordosis
in response to
mounting
newborn
female

8. Process of Sexual Differentiation:
MALES
FEMALES
masculinized
defeminized
feminized
demasculinized
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
“active”

9. 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

10. 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

11. 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

12. 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
guinea pigs/primates
dependent on
aromatization
male sex
behavior
not dependent on
aromatization

13. Species Differences:
There are species differences in what processes are masculinized and defeminized.
male rats
male primates
male sex
behavior
Masculinized:
true
Defeminized:
no positive
feedback response
to estrogen; no
support of ovulation
not true

14. 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

15. 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

16. 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

17. 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
Ventricular
Zone
day 18
day 19
Developing
Brain
[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

18. Hormones--Target-Derived Neurotrophic Function:
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
Perineal
muscles
during
critical period
of development,
muscles can bind
androgens while the
motoneurons
cannot
secrete a
“retrograde factor”
that leads to survival
of neurons
SNB

19. Example Question:
What structures within the nervous system would be masculinized or feminized in a male rat with testicular feminization mutation?
SDN-POA
SNB
normal
male
masculinized
(large)
masculinized
(large)
male with
TFM

20. 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)
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

21. 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

22. 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