Reproductive Aging
37
(a review of general endocrinology)
• Overview of the neuroendocrine
control of reproduction: E2 - and +
feedbacks, preovulatory GnRH and
LH release, preovulatory surge of
LH and ovulation
• Reproductive aging (menopause)
is brought about by a decline in the
activation of the GnRH system to
the positive feedback of E2 to
induce preovulatory surges of LH
?
S
• Reproductive failure associated
with aging is linked to disfunction
of hypothalamic clock genes
E
Today’s lecture
control and “story lines”
?
S
E
1
Take Home Message (THM)
The menopause marks the end of a woman's reproductive life. During the postmenopausal period, plasma estrogen concentrations
decrease dramatically and remain low for the rest of her life, unless she chooses to take hormone replacement therapy. During the past 20
years, we have learned that changes in the central nervous system are associated with and may influence the timing of the menopause in
women. Recently, it has become clear that estrogens act on more than just the hypothalamus, pituitary, ovary, and other reproductive
organs. In fact, they play roles in a wide variety of nonreproductive functions. With the increasing life span of humans from approximately 50
to 80 years and the relatively fixed age of the menopause, a larger number of women will spend over one third of their lives in the
postmenopausal state. It is not surprising that interest has increased in factors that govern the timing of the menopause and the
repercussions of the lack of estrogen on multiple aspects of women's health. We have used animal models to better understand the complex
interactions between the ovary and the brain that lead to the menopause and the repercussions of the hypoestrogenic state.
Our results show that when rats reach middle age, the patterns and synchrony of multiple neurochemical events that are critical to the preovulatory GnRH surge undergo subtle changes. The
precision of rhythmic pattern of neurotransmitter dynamics depends on the presence of estradiol.
Responsiveness to this hormone decreases in middle-aged rats. The lack of precision in the
coordination in the output of neural signals leads to a delay and attenuation of the LH surge, which
lead to irregular estrous cyclicity and, ultimately, to the cessation of reproductive cycles. We also have examined the impact of the lack of estrogen on the vulnerability of the brain to injury. Our work establishes that the absence of
estradiol increases the extent of cell death after stroke-like injury and that treatment with low physiological levels of estradiol are profoundly
neuroprotective. We have begun to explore the cellular and molecular mechanisms that underlie this novel nonreproductive action of
estrogens. In summary, our studies show that age-related changes in the ability of estradiol to coordinate the neuroendocrine events that
lead to regular preovulatory GnRH surges contribute to the onset of irregular estrous cycles and eventually to acyclicity. Furthermore, we
have shown that the lack of estradiol increases the vulnerability of the brain to injury and neurodegeneration.
Neuroendocrine modulation and repercussions of female reproductive aging.
PM Wise, MJ Smith, DB Dubal, ME Wilson, SW Rau, AB Cashion, M Bottner and KL Rosewell, Dept of
Physiology, College of Medicine, Lexington, Kentucky 40536-0208. The Endocrine Society (2002).
GnRH and the HPG axis
GnRH
FSH & LH
2
GnRH and the HPG axis
(the release of GnRH occurs in a pulsatile fashion)
LH/FSH
GnRH
LH
from POA
to the ME
E2
FSH
P4
GnRH and the HPG axis
Tonic and phasic control of GnRH
•
sensors or feedback receptors detect a
physiological variable under control (E2/P4)
•
the sensor links with an integration center to
compare the variable under control against a
system set point or command signal (CS)
•
•
•
thus, integration center gets afferent information
and communicates with effectors via efferent
pathways (GnRH)
the integration center output signal is amplified
in the efferent pathway leading to the effectors
the variable under control reflects the function
of the effector system and is used as a negative
feedback control in the tonic control or as a
positive feedback control in the phasic control.
CS
E2
integrator
P4
receptor
GnRH
AP
gonad
E2 / P4
3
GnRH and the HPG axis
multiple inputs
The tonic control
set
point
E2
single / limited
output
dx
dt
A set point can be modulated by its own inputs.
An integrator or “comparator”, compares what
it should be (set point) against what actually is
(the negative feedback signal).
LH
S
-Fb
ME
E
the tonic control responds to the
first derivative of plasma estradiol
time in hours
GnRH and the HPG axis
multiple inputs
The phasic control
set
point
E2
single / limited
output
A set point can be modulated by its own inputs.
An integrator or “comparator”, compares what
it should be (set point) against what actually is
(the negative feedback signal).
LH
S
-Fb
+Fb
POA
E
the phasic control responds to the
second derivative of plasma estradiol
time in hours
4
GnRH and the HPG axis
Knobil et al, Endocrinology (1973).
The phasic control
E2
E2
LH
LH
time in hours
-Fb
+Fb
POA
the phasic control responds to the
second derivative of plasma estradiol
time in hours
Reproductive Aging
• a hallmark of the
human
postmenopausal
state is the total
exhaustion of
ovarian follicles.
• in rodent models,
which also exhibit
reproductive
senescence, it is
less clear whether
the follicular pool is
totally exhausted.
However, the
number of follicles
remaining in the
ovary is minimal.
LHRH
output
1
2
3
4
(less E2 is needed to induce estrous
behavior than to induce an LH surge)
E2
output
day of
estrus
1 regular cycles
2 variable cycles
3 constant estrus
4 constant diestrus
5
Reproductive Aging
1
1 regular cycles
2 variable cycles
3 constant estrus
4 constant diestrus
2
3
4
(less E2 is needed to induce estrous
behavior than to induce an LH surge)
Neuroendocrine modulation and repercussions of female reproductive aging.
PM Wise, MJ Smith, DB Dubal, ME Wilson, SW Rau, AB Cashion, M Bottner and KL Rosewell, Dept of
Physiology, College of Medicine, Lexington, Kentucky 40536-0208. The Endocrine Society (2002).
Reproductive Aging
LHRH=GnRH
A reduced proportion of luteinizing hormone (LH)-releasing hormone neurons express Fos
protein during the preovulatory or steroid-induced LH surge in middle-aged rats.
Rubin BS, Lee CE and King JC. Department of Anatomy and Cell Biology, Tufts University School of
Medicine, Boston, MA 02111.
Biology of Reproduction 51:1264-1272, 1994.
6
Reproductive Aging
Young
LHRH=GnRH
MiddleAged
Reconstructions of populations of LHRH neurons in young and middle-aged rats reveal
progressive increases in subgroups expressing Fos protein on proestrus and age-related
deficits. Rubin BS, Mitchel S, Lee CE and King JC. Department of Anatomy and Cell Biology, Tufts
University School of Medicine, Boston, MA 02111.
Endocrinology 136 (9) 3823-3830,1995.
Reproductive Aging
LHRH=GnRH
Results of previous studies suggest that altered patterns of LHRH neurosecretion contribute to
attenuated LH surges and the eventual cessation of ovulation in aging female rats. The present
study compared evidence of LHRH neuronal activation in conjunction with the preovulatory and
steroid-induced LH surge in young and middle-aged animals to determine whether age-related
alterations could be detected. Double immunocytochemical protocols were used to colocalize
LHRH and the protein product of the proto-oncogene c-fos, which increases within the nucleus of
LHRH neurons in association with spontaneous or induced LH surges. The mean proportion of
LHRH neurons containing immunoreactive Fos was higher in the brains of young compared to
middle-aged females in association with both the preovulatory (p < 0.01) and the steroid-induced
LH surge (p < 0.001). The time course of activation of LHRH neurons was delayed in the brains of
aging females, and the proportion of double-labeled LHRH neurons remained elevated longer in the
brains of young compared to middle-aged steroid-treated females. Moreover, regional differences in
LHRH neuronal activation were observed both within and between age groups. The data presented
suggest that reduced LHRH neuronal activation may contribute to the attenuation and eventual loss
of preovulatory LH surges in middle-aged female rats.
A reduced proportion of luteinizing hormone (LH)-releasing hormone neurons express Fos
protein during the preovulatory or steroid-induced LH surge in middle-aged rats.
Rubin BS, Lee CE and King JC. Department of Anatomy and Cell Biology, Tufts University School of
Medicine, Boston, MA 02111.
Biology of Reproduction 51:1264-1272, 1994.
7
Reproductive Aging
LHRH mRNA levels were examined in young and middle-aged female rats at 4 times (10:00 h,
14:00 h, 18:00 h and 20:00 h) on the day of a steroid-induced LH surge by in situ hybridization with
a digoxigenin-labeled riboprobe. Young, but not middle-aged females, exhibited dynamic temporal
changes in the number of LHRH mRNA positive neurons detected in the organum vasculosum of
the lamina terminalis-preoptic area (OVLT-POA) continuum. Specifically, fewer LHRH mRNA
positive neurons were detected at 18:00 h compared with the number detected at 14:00 h and
20:00 h (P < 0.01) in the OVLT-POA of young females. All LHRH mRNA positive neurons present
in 4 anatomically matched sections through the rostral POA of young and middle-aged animals
were digitized for detailed computer-assisted analysis of the hybridization reaction product. The
mean hybridization area (P < 0.00025) and integrated optical density per cell (P < 0.006) were
reduced in middle-aged compared to young females consistent with a relative age-related decline
in LHRH mRNA levels. Moreover, an age-related reduction in cellular and/or regional hybridization
area was noted at each of the time points examined (P < 0.05-P < 0.001). These data confirm
earlier reports of dynamic changes in LHRH mRNA levels on the day of an LH surge. Furthermore,
they support a role for age-related alterations in LHRH gene expression in the disruption of regular
estrous cyclicity in middle-aged females.
Luteinizing hormone-releasing hormone gene expression differs in young and middle-aged
females on the day of a steroid-induced LH surge.
Rubin BS, Lee CE, Othomo M and King JC. Department of Anatomy and Cell Biology, Tufts
University School of Medicine, Boston, MA 02111.
Brain Research 770 (1-2):267-276, 1997.
Reproductive Aging
LHRH=GnRH
Fos expression has been used as a marker of activation of neuroendocrine cells including LHRH
neurons. In this study, Fos protein was localized within LHRH neurons in young and middle-aged rats to
trace the temporal and spatial pattern of LHRH neuronal activation associated with the preovulatory LH
surge. Animals were killed during the late morning, afternoon, and evening of proestrus. Dual
immunocytochemical protocols localized LHRH and LHRH/Fos neurons, and computer-assisted
methods were used to reconstruct forebrain populations of single- and double-labeled LHRH neurons.
Although a significant increase in the number of LHRH/Fos neurons was noted by evening in both age
groups, a greater increase was observed in young (12% in morning, 28% in afternoon, and 62% by
evening) compared with aging females (5% in morning, 10% in afternoon, and 40% by evening).
Reconstructions of LHRH and LHRH/Fos neurons revealed time- and age-dependent differences in Fos
expression within LHRH neurons. In young females, LHRH/Fos neurons were restricted to central
regions of the population of LHRH neurons on the morning of proestrus. By evening, Fos expression
was also observed in more peripheral and caudal LHRH neurons. In middle-aged females, Fos
expression was restricted to ventral subgroups of LHRH neurons on the afternoon of proestrus. By
evening, more LHRH neurons contained Fos protein, however, few were located in the dorsal aspect of
the population. These data trace the progressive increase in activation of LHRH neurons during the
preovulatory LH surge in young females and reveal deficits in this pattern of activation by middle age.
Reconstructions of populations of LHRH neurons in young and middle-aged rats reveal
progressive increases in subgroups expressing Fos protein on proestrus and age-related
deficits. Rubin BS, Mitchel S, Lee CE and King JC. Department of Anatomy and Cell Biology, Tufts
University School of Medicine, Boston, MA 02111.
Endocrinology 136 (9) 3823-3830,1995.
8
Reproductive Aging
%
GnRH
and VIP
neurons
with
cFos
%
GnRH
and VIP
neurons
without
cFos
SCN intact
(have repro rhythm)
untransfected
vehicle alone
SCN lesioned
(no repro rhythm)
transfected with clock delta 19
time in hours
So, … reproductive aging might be a disfunction
of the neuroendocrine control associated with clock genes
Clock genes & reproduction
Circadian gene expression regulates pulsatile GnRH secretory pattern in the hypothalamic GnRH
secreting GT 1-7 cell line. PE Chappell, RS White and PL Mellon, Dept. Reproductive Medicine, University of California, San Diego, La
J of Neuroscience 23: (35), 1202-1213,2003.
Jolla, CA.
9
Circadian gene expression regulates pulsatile GnRH secretory pattern in the hypothalamic GT1-7 cell line
B Bmall1
C Clock
(+)
(-)
P
C
CKI
Per P
Cry C
nucleus
phosphorylation
degradation
cytoplasm
confirm expression of clock genes
In the hypothalamic GT1-7 cell line
Circadian gene expression regulates pulsatile GnRH secretory pattern in the hypothalamic GT1-7 cell line
mRNA protection assay shows mRNA
oscillations after shift to SF media
and FSK of mPer1,2 /mCry1,2. No
oscillations are present in absence of
perturbation (SF or FSK)
This and other labs have shown that:
cAMP and MAPK pathways are
involved in regulating circadian gene
expression, GnRH transcription and
GnRH secretion
cAMP is involved in the generation of
the pulse rhythm of GnRH secretion
transient increases in mPer1 mRNA
levels in GT1-7 cells after FSK
FSK SS IM PMA SNP
correlate with increases in GnRH
gene
products
are
capable
of
exhibiting cyclic
secretion in static cultures
mRNA accumulation rhythms in the GT1-7 cells
10
Circadian gene expression regulates pulsatile GnRH secretory pattern in the hypothalamic GT1-7 cell line
protein levels of Bmall oscillate in
GT1-7 cells after a serum shock (panel
A)
the transcription factor Bmall binds with
its heterodimeric partner, Clock, to
increase trancription of Cry and Per
genes, forming the “positive limb” of
the circadian clock feedback loop
Increase in Clock:Bmall heterodimers
binding to mPer1 promoter E-boxes
increases transcription of mPer1 gene
protein levels of mPer1 in GT1-7 nuclear
extracts also cycle, exhibiting peaks
8-12hr out of phase with Bmall protein a functional clock may exist in GT1-7 cells
levels (panel B)
Circadian gene expression regulates pulsatile GnRH secretory pattern in the hypothalamic GT1-7 cell line
GT1-7 cells transiently transfected with
mPer1-luciferase (the TK-ß-galactosidase
plasmid was used as an internal control).
The mPer1-luciferase gene expression
oscillates with a 20-24hr period in serum
free (A) and serum-shocked conditions (B)
co-transfection with plasmid expressing a
dominant negative Clock gene, Clock-19,
severely blunted mPer1-luciferase cycling
in GT1-7 cells (in both A, B)
serum free conditions
serum - shocked conditions
co-transfection with empty control vector,
pcDNA3.1, did not affect these oscillations
transient over-expression of mCry1, a
potent repressor of mPer1 promoter,
reduced 20-50% of control oscillations
circadian clock maintains transcriptional
regulatory relationships in GT1-7 cell line
11
Circadian gene expression regulates pulsatile GnRH secretory pattern in the hypothalamic GT1-7 cell line
GFP fluoresence in GT1-7 cells grown on
beads after 48hr of perifusion. GT1-7 cells
were transfected with either a rat 5’-GnRHeGFP (18-36% efficiency) or the parent
CMV-eGFP vector (25-60% efficiency) and
perifused for 48hr
Panel A: bright field micrograph of a
cluster of GT1-7 cells on Cytodex beads
after 24hr of static incubation in DMEM /
10% FCS followed by 48hr of perifusion
with KRB
Panel B: Fluoresence imaging revels
robust GFP expression in the same cell
cluster 72 hr after transfection and
perifusion
GFP fluorescence revels robust transgene
expression in GT1-7 cells grown in beads
Circadian gene expression regulates pulsatile GnRH secretory pattern in the hypothalamic GT1-7 cell line
robust GFP
fluorescence
levels after
transgene
expression in
GT1-7 cells
grown in
beads (fig 5B)
dominant negative
Clock-19 gene
blunts mPer1luciferase cycling
in GT1-7 cells, but
the empty control
vector pcDNA3.1
does not (fig 4B)
perturbation of circadian clock by Clock19 transfection disrupts GnRH release
12
Circadian gene expression regulates pulsatile GnRH secretory pattern in the hypothalamic GT1-7 cell line
robust GFP
fluorescence
levels after
transgene
expression in
GT1-7 cells
grown in
beads (fig 5B)
dominant negative
Clock-19 gene
blunts mPer1luciferase cycling
in GT1-7 cells, but
the empty control
vector pcDNA3.1
does not (fig 4B)
perturbation of circadian clock by Clock19 transfection decreases the frequency
of GnRH pulsatile release in GT1-7 cells
Circadian gene expression regulates pulsatile GnRH secretory pattern in the hypothalamic GT1-7 cell line
robust GFP
fluorescence
levels after
transgene
expression in
GT1-7 cells
grown in
beads (fig 5B)
transient overexpression of
mCry1, a potent
repressor of mPer1
promoter, reduced
20-50% of control
oscillations (fig 4A)
overexpression of mCry increases GnRH
pulse amplitude in GT1-7 transfected cells
13
Circadian gene expression regulates pulsatile GnRH secretory pattern in the hypothalamic GT1-7 cell line
circadian clock function is linked to the secretory machinery
governing timed GnRH pulse release from GT1-7 cells in
culture, a link that appears to be preserved in vivo
Clock genes & reproduction
The GT1-7 molecular clock may be coupled to
pulsatile GnRH secretion
Clock-19
nucleus cytoplasm
Cycling levels of Per and Cry are required for
GnRH release pattern
Clock-19 lowers pulse frequency & increases
pulse amplitude variability
Cry overexpression increases GnRH pulse
amplitude (other functions ??)
Role of Clock-19 on Cry & of Cry as
Clock:Bmall transactivation inhibitor
Role of Clock:Bmall on E-box motifs of “clock
controlled genes” (CCG)
Pulsatile GnRH after an LH surge might depend
on clock function
B Bmall1
C Clock
(+)
(-)
P
C
CKI
Per P
Cry C
phosphorylation
degradation
Cry overexpression
Circadian gene expression regulates pulsatile GnRH secretory pattern in the hypothalamic GnRH
secreting GT 1-7 cell line. PE Chappell, RS White and PL Mellon, Dept. Reproductive Medicine, University of California, San Diego, La
J of Neuroscience 23: (35), 1202-1213,2003.
Jolla, CA.
14