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Floxing around with the clock: The effects of circadian clock gene deletion on the neuroendocrine control of reproduction. Karen J. Tonsfeldt Patrick Chappell, PhD. OSU Dept. of Zoology. ♀. Follicle growth Estrogen and progesterone in ovaries Ovulation.

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slide1

Floxing around with the clock: The effects of circadian clock gene deletion on the neuroendocrine control of reproduction

Karen J. Tonsfeldt

Patrick Chappell, PhD.

OSU Dept. of Zoology

overview gonadotropin releasing hormone gnrh

  • Follicle growth
  • Estrogen and progesterone in ovaries
  • Ovulation
Overview: Gonadotropin Releasing Hormone (GnRH)

Brain

Timing cues

Hypothalamus

GnRH

Estrogen

Pituitary

LH & FSH

Gonads

overview gnrh release
Overview: GnRH Release

Maintenance of fertility

Ovulation

Infertile

Hypothalamus

GnRH

Pituitary

LH & FSH

Gonads

overview circadian clock

CRY

CRY

CRY

CRY

CRY

CRY

CRY

CRY

CRY

Overview: Circadian Clock

Nucleus

Cytoplasm

Bmal1

BMAL

BMAL

BMAL

BMAL

BMAL

BMAL

BMAL

Clock

PER

PER

PER

PER

PER

PER

PER

PER

PER

CLOCK

CLOCK

CLOCK

CLOCK

CLOCK

CLOCK

CLOCK

Period

Cryptochrome

circadian clock manipulations activity

PER

BMAL

CLOCK

CRY

Circadian Clock Manipulations: Activity

Wild Type

Bmal1

Clock

Clock Mutants

Bmal Knockouts

Period

Cryptochrome

Vitaterna et al., Science 94; McDearmon et al., Science 06

competing theories
Competing Theories

GnRH controlled by SCN

GnRH controlled by independent internal clocks

SCN

  • Requires functional clock in GnRH neurons, no SCN
  • Would require a fully functional SCN
cre lox system
Cre/Lox System

Promoter

Cre

loxP

Red

loxP

loxP

loxP

loxP

loxP

loxP

loxP

loxP

loxP

loxP

loxP

R

R

R

R

R

R

R

R

R

R

loxP

loxP

loxP

loxP

loxP

loxP

loxP

loxP

loxP

loxP

NSP1

Cre

loxP

Red

loxP

NSP1

loxP

Red

Target

loxP

Cre

loxP

loxP

Red

loxP

loxP

Cre

Heterozygote: Cre+, LoxP+/-

Homozygote: Cre+,LoxP+/+

WT: Cre+ or -, LoxP-/-

cre lox system the bsd cre bmal flox
Cre/Lox System: The BSD Cre/Bmal Flox

BSD

Cre

loxP

Bmal

loxP

Brain Specific Driver

Cre

loxP

Bmal

loxP

Takahashi

Takahashi

BSD Cre / Bmal Flox

Bmal

Fertile!

Takahashi

cre lox system the gnrh cre bmal flox
Cre/Lox System: The GnRH Cre/Bmal Flox

GnRH

Cre

loxP

Bmal

loxP

GnRH

Cre

loxP

Bmal

loxP

Levine

Takahashi

GnRH Cre / Bmal Flox

Bmal

Chappell

current progress
Current Progress

BSD Cre / Bmal Flox

GnRH Cre / Bmal Flox

Wild Type

  • Develop colony
  • Determine success of Cre/Lox
  • Characterize reproduction
  • Characterize coexpression of Bmal and GnRH
  • Determine success of Bmal KO in GnRH neurons

Bmal

Bmal

bsd cre bmal flox
BSD Cre / Bmal Flox

Stained for Bmal & DAPI

Bmal

Homozygote

Heterozygote

Wild Type

slide13

BSD Cre / Bmal Flox

WT stained for GnRH & BSD

GnRH

BSD

Bmal

slide14

BSD Cre / Bmal Flox

Stained for GnRH & BSD

GnRH

BSD

Bmal

bsd cre bmal flox progress
BSD Cre / Bmal Flox Progress
  • Conclusions:
  • Functioning SCN not needed for fertility
  • Future work:
  • - Characterize estrous cycle

Bmal

gnrh cre bmal flox
GnRH Cre / Bmal Flox

Establishing the colony:

Flox

Het

WT

Cre-

Cre+

Bmal

mating frequency
Mating Frequency

Fecundity

Litter size

Gestation

% Males

Bmal

immunohistochemistry
Immunohistochemistry

Stained for GnRH and Bmal

Bmal

immunohistochemistry19
Immunohistochemistry

Stained for GnRH and Bmal

Bmal

gnrh cre bmal flox progress
GnRH Cre / Bmal Flox Progress
  • Conclusions:
  • Functioning clock in GnRH neurons not necessary for fertility
  • Future work:
  • Characterize estrous cycle
  • Characterize GnRH release
  • Expand breeding pair results

Bmal

slide21
WTs

Stained for GnRH & Bmal

wt progress
WT Progress
  • Conclusions:
  • GnRH neurons do not always express Bmal
  • Future work:
  • Further characterization
conclusions
Conclusions

Functional GnRH clocks may also not be necessary for fertility

Bmal

Bmal

Bmal and GnRH are not always co-localized

A functional SCN is not necessary for fertility

acknowledgments
Acknowledgments

Mus musculus

Howard Hughes Medical Institute Fellowship

OSU:

Chappell lab: Patrick Chappell, Cheri Goodall, Kristin Latham, Chris Leonard

Northwestern University:

Levine lab: Jon Levine, Maricedes Acosta-Martinez, Brigitte Mann, Claire Capshew

Takahashi lab: Joe Takahashi, Tina Pejchal

Funding:

K01 DK064919 and R01 DK073571 to PEC, URISC & HHMI to KJT