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

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

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  1. 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

  2. ♀ • Follicle growth • Estrogen and progesterone in ovaries • Ovulation Overview: Gonadotropin Releasing Hormone (GnRH) Brain Timing cues Hypothalamus GnRH Estrogen Pituitary LH & FSH Gonads

  3. Overview: GnRH Release Maintenance of fertility Ovulation Infertile Hypothalamus GnRH Pituitary LH & FSH Gonads

  4. 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

  5. Overview: Suprachiasmatic Nucleus

  6. 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

  7. 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

  8. 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-/-

  9. 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

  10. 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

  11. 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

  12. BSD Cre / Bmal Flox Stained for Bmal & DAPI Bmal Homozygote Heterozygote Wild Type

  13. BSD Cre / Bmal Flox WT stained for GnRH & BSD GnRH BSD Bmal

  14. BSD Cre / Bmal Flox Stained for GnRH & BSD GnRH BSD Bmal

  15. BSD Cre / Bmal Flox Progress • Conclusions: • Functioning SCN not needed for fertility • Future work: • - Characterize estrous cycle Bmal

  16. GnRH Cre / Bmal Flox Establishing the colony: Flox Het WT Cre- Cre+ Bmal

  17. Mating Frequency Fecundity Litter size Gestation % Males Bmal

  18. Immunohistochemistry Stained for GnRH and Bmal Bmal

  19. Immunohistochemistry Stained for GnRH and Bmal Bmal

  20. 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

  21. WTs Stained for GnRH & Bmal

  22. WT Progress • Conclusions: • GnRH neurons do not always express Bmal • Future work: • Further characterization

  23. 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

  24. 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

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