Victoria Grimwood1 , Patrick Chappell2
Department of Bioresource Research, Oregon State
University, Corvallis OR1
Department of Veterinary Medicine, Oregon State
University, Corvallis OR2
Hypothalamic-pituitary-gonadal (HPG) axis regulation
+
Hypothalamus
Pulse vs. Surge
GnRH
Anterior
Pituitary
LH
Steroid
Hormones
GnRH Neurons
 Location
 Approximately 2000 in hypothalamus
 Scattered cell bodies
 Axons release neuropeptide outside of brain
 Pulsatile secretions
 Possess many receptors
Suprachiasmatic nuclei of the
hypothalamus (SCN)
 Pacemaker clock
 Activated by light
 Coordination peripheral clocks
 Secretes neuropeptides
 Composed of heterogeneous mixture of cell types
 Oscillation at molecular, cellular, and tissue levels
Kisspeptin
 Secreted by Kiss-1 neurons
 Powerful stimulator GnRH release
 Essential to pubertal progression and feedback to steroid
hormones
 May be essential to GnRH pulsatile secretions
When pulse comes to surge:
Do endogenous oscillators play a role in modulation of
GnRH secretion patterns in response to estradiol?
SCN
DNS
GnRH
Kiss1?
When pulse comes to surge:
Do endogenous oscillators play a role in modulation of
GnRH secretion patterns in response to estrogen?
Proestrus
DNS
SCN
E2
GnRH
Kiss1?
•We use our in vitro GnRH cell models (GT1-7 cells) to examine direct
dose- and time-dependent effects of E2 as well as circadian genes.
Gpr54/Kiss1-R expression in GT1-7 cells is
rhythmic in the presence of 17β-E2
100pM E2
2
1
0
Fold change from 0h
3
EtOH
0
12
24
36
48
0
hours
12
24
36
48
Circadian Rhythms
 Biological process containing an endogenous
approximately twenty-four hour oscillation pattern
 Regulates many bodily functions
 Sleep-wake cycle
 Hormonal secretion
 Core body temp, hunger, heart rate, ect.
 Occur throughout many taxonomic levels
 Prokaryotes, basic eukaryotes, and multicellular organisms
 All organisms have highly conserved clock
Molecular Clock
 Coordinate circadian feedback
 Can be entrained by extracellular signaling
 Initiate intracellular response
 Regulate transcription patterns of specific genes
 Clock, Bmal1, Period, and Cryptochrome
 In response to cell signaling
bHLH-PAS txn factors
Period
Cryptochrome
BMAL1
CLOCK
B Cl
E-box
CCG
OUTPUT
B Cl
P Cy
E-box
Per1/Per2
P
E-box
Cry1/Cry2
Cy Cy
P
B Cl
Nuc
Cyto
Gpr54-luciferase expression is significantly higher when
co-transfected with CLOCK/BMAL1
average fold induction compared to
GPR54-luc + pcDNA3.1
*
luciferase fold induction
relative luciferase
40
30
20
10
0
pcDNA3.1
6
4
2
0
CLOCK/BMAL1
B Cl
?
GPR54 promoter
8
GPR54-luc + CLOCK/BMAL1
luciferase
LUC
80
80
70
70
relative luciferase
relative luciferase
Gpr54-luciferase expression is depressed when treated with
E2, even when co-transfected with CLOCK/BMAL1
60
20
10
0
60
20
10
0
mock
E2
GPR54-luc+ pcDNA3.1
GPR54-luc + CLOCK/BMAL1
E2
B Cl
?
GPR54 promoter
mock
E2
mock
luciferase
E2
mock
LUC
E2
Estrogen Receptors (ER)
 Nuclear hormone family of intracellular receptors
 Multiple isoforms of estrogen receptors exist


Estrogen receptor alpha (ERα)
Estrogen receptor beta (ERβ)
A.
B.
C.
D.
E.
Legend
Estrogen receptor
Estrogen
Estrogen helper
proteins
Cell nucleus
DNA
Estrogen Receptor Alpha
 Believed essential to fertility
 Prior study demonstrated that E2 in ERα breast cancer
cell lines induces Per2 mRNA levels in mammary
epithelial cells
 Physical interaction between Per2 and ERα
 Why we looked at ERβ
 Not expressed by GnRH neurons
Estrogen Receptor Beta
 Present in GnRH neurons
 E2 binding appears to down-regulate Erβ expression
 Negative feedback mechanism
 E2 possibly interacts with endogenous circadian
oscillators at ERβ
We believe direct protein-protein
interactions exist between clock component
BMAL1 and ERβ in GT1-7 and SCN cell lines,
modulated by presence of E2
?
B Cl
E-box
-steroid hormone modulation of the
cellular clock
GPR54
GPR54
E2
ERβ?
B Cl
E-box
B Cl
E-box
Per1/Per2
P
?
Cry1/Cry2
Cy
B Cl
 identify protein-protein interactions
between ERβ and BMAL1 in multiple in
the absence and presence of E2
Nuc
Materials and Methods
Maintanence of Cell Lines
-GT1-7, E2 GT1-7, SCN Cell Lines
-Immortalized neuronal cell line
Protein extraction and quantification
-Cell lysis and protein extraction
-Quantification BCA Assay
Co-Immunoprecipitation
-isolate specific protein and complexes with direct
protein-antibody interactions
Western Blot
-Identify protein by size
-Anti-estrogen receptor beta primary antibody
(rabbit)
-Anti-BMAL1 primary antibody (rabbit)
-Anti-rabbit secondary antibody
Co-Immunoprecipitation
 Primary antibody coupled to




column
Pre-cleared lysate in control
column
Lysate incubated in antibody
coupled column
Wash of unbound protein
Elution of protein complex
associated with column
Western Blot
 Western blot utilized to produced visual results of Co-
Immunoprecipition
 Run products of Co-IP through polyacrylamide gel
 Bind products with target primary and secondary
antibodies
SCN 2.2 Co-IP Blot
 Bands perceived in antibody verification, unbound protein,
elution, and SCN protein lanes
 Bands only appeared just above 50kDa in the elution lanes.
GT1-7 Co-IP Blot
 Bands in antibody verification, unbound protein, elution, and GT1-7 lanes
 Elution bands at ~53kDa in each
 Unexpected
 Fewer bands in BMAL coupled blot
GT1-7 BMAL1 coupled, Erβ probed
 Only blot supporting hypothesis
 Evidence of Erβ protein in elution lanes
 Theoretically contain only protein associated with antiBMAL1 antibody
 Band at 53kb appears in each lane except negative control
and blank

Expected
Estradiol exposed GT1-7 Co-IP Blot
 Both blots exhibit lack of association in elution lanes
 BMAL1 probed possesses more bands in protein and unbound protein
lanes
 Unexpected
Estradiol Modulation
 Estradiol down-regulates positive clock elements
 BMAL1 is positive clock transcription factor
 No indication of BMAL1-Erβ protein interactions
 Strong band in lysate lanes at 53kDa
 Should see 53kDa in Erβ probed elution lane
Original Hypothesis
 Direct protein-protein interactions between BMAL1 and
Erβ in GT1-7 (in presence or absence of estradiol) and
SCNcell lines appear unclear
 Positive Erβ and BMAL1 bands expressed in lysate did not
show in altogher appropriate positions of elution
 Only bands of roughly Erβ size appeared in elution lanes
 Confirmatory band in BMAL1 coupled column when probed
with ERβ
 Generation of an effective BMAL1or ER beta antibody in
species other than rabbit necessary
Future Research
 Chromatin Immunoprecipitation (chIP)
 Determines whether a specific genomic region interacts
with the protein of interest
 Transcription factors on promoters or other DNA binding
sites
 If promoter known, can determine if ERbeta and BMAL1
binding site close enough for direct contact


where on the Kiss1R promoter ERbeta and BMAL1 are binding
Domain(s) of each transcription factor is/are required for this
interaction
Kiss-1R Promoter
 E2 response elements (ERE) and E-boxes within the
GPR54 promoter sequence
 CLOCK/BMAL1 suspected binding sites
Infertility
 “Circadian health” essential
to fertility
 Proper rhythmic cycling
necessary for ovulation
 Clock disregulation could
cause missed surges
 Less ovulation
 Decreasing fertility rate
since Industrial Revolution
 “Abnormal” sleep wake
cycles
 Swing shift workers

Nurses, flight attendents
• Increase of hormone dependent cancers
• Breast and Prostate cancers combine to roughly 30% of total new
cancer cases each year
-American Cancer Association
Acknowledgements
 The Chappell Lab
 Dr. Patrick Chappell
 Cheri Goodall
 Ian Hilgart, Briana Knight, Robinson Taylor, Alex Koosman,
Kristen Tonsfeld
 Wanda Crannell
 Funding
 ER Jackman Grant
 Honors Experience Award