Generating kisspeptin cell lines to investigate their role in reproduction

Transcription

1 Generating kisspeptin cell lines to investigate their role in reproduction Dakota C. Jacobs 1 Jadwiga M. Giebultowicz 2, and Patrick E. Chappell 3 1 Bioresource Research, 2 Department of Integrative Biology, College of Science, 3 Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, OR

2 Neuroendocrinology Intercommunication between the nervous system and the endocrine system Allows hypothalamic control of homeostasis Pituitary Gonads Thyroid Kidneys Pancreas Hypothalamus

3 Testosterone HPG Axis LH/FSH Testes GnRH = gonadotropin releasing hormone Hypothalamus GnRH Pituitary LH/FSH Ovaries LH = luteinizing hormone FSH = follicle stimulating hormone Estrogen

4 Understanding Regulation of GnRH Secretion is Changing

5 GnRH = gonadotropin releasing hormone GnRH Neurons AVPV Arc ME

6 Significance of Kiss1 and Kiss1R GnRH stimulation by kisspeptin is required for puberty onset Dramatic shifts in hormone balance Non-uniform body growth Purpose of puberty is to attain sexual maturity Pubertal onset fails to initiate without functional Kiss1/Kiss1R system

7 Kisspeptin-Releasing Neurons Kisspeptin stimulates GnRH neurons to secrete GnRH Kisspeptin neurons are sensitive to estrogen Estrogen receptor alpha (ERα) Endocr Rev Oct;30(6):713-43

8 Kisspeptin (Kiss1) Secretion Profile AVPV and Arc neurons do not respond to E 2 the same way AVPV + E 2 Increase in Kiss1 Endocr Rev Oct;30(6): Arc + E 2 Decrease in Kiss1

9 Goals of this Project Isolate AVPV and Arc neurons from a mouse model? Culture neurons Generate an in vitro model of AVPV and Arc neurons using third generation lentiviral packaging system Endocr Rev Oct;30(6):713-43

10 Methodology Transgenic mouse model; GFP-tagged kiss1 in AVPV and arcuate nuclei Sacrifice adult female mice Isolate AVPV and Arc hypothalamic regions using a vibratome

11 Vibratome Procedure GFP+ Brain ACSF Metal Platform Agar Block Agar Block AVPV Arc AVPV = 500 µm Arc = 1000 µm

12 Methodology Suspend isolated explants in cell culture media with collagenase Shake at 200 rpm at 37 C for 45 minutes Add fetal bovine serum (FBS) to deactivate collagenase

13 Methodology AVPV Arc Plate AVPV and Arc samples in 24 well plate Suspend samples in 20% FBS DMEM with penicillin, streptomycin, and gentamicin

14 AVPV/Arc Primary Culture AVPV Arc

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16 Fluorescence-Activated Cell Sorting (FACS) Procedure Sorts neurons based on fluorescence Re-suspend AVPV and Arc samples in PBS Sort into 20% FBS DMEM Re-plate purified samples

17 FACS Mechanism Suspended AVPV or Arc cells Sorts GFP +- cells separate from GFP - cells Face scatter = granularity Side scatter = size AVPV and Arc samples are run separately Fluorescence Face scatter/side scatter Single-file cells Laser source GFP + GFP -

18 FACS Results ARC AVPV Viable Neurons Flow cytometry follows same pattern over several sorts

19 Purified AVPV and Arc Cultures AVPV Arc

20 Third Generation Lentiviral Packaging System Reduced HIV-1 genome to four components to eliminate virulence 1) Two packaging vectors 2) Envelope vector 3) Transgene vector Transfection HEK 293T Cells Desired Effect Infection of Target Cells

21 VIF VPU NEF LTR GAG PRO POL ENV LTR TAT TAT REV REV Promoter GAG POL Promoter REV Promoter ENV LTR Promoter TA-g LTR

22 Immortalization Method Transfect HEK 293T with four plasmids 1) SV40 large T-antigen (TAg) Transgene Plasmid 2) prev packaging plasmid 3) pgag/pol packaging plasmid 4) pvsv-g envelope plasmid Intracellular virus assembly Infect AVPV and Arc neurons Large T-antigen cdna Packaging Plasmids Envelope

23 Immortalization Results Infected AVPV and Arc kiss1-expressing neurons begin proliferating after 4 weeks post-infection AVPV Arc

24 Results AVPV and Arc neurons retain unique estrogen treatment-induced expression patterns AVPV Arc AVPV kiss1 expression increases after 20 minute exposure of E 2 Arcuate kiss1 expression decreases after 4 hour exposure of E 2 AVPV No E 2 Arc No E 2 AVPV + E 2 Arc + E 2

25 Limitations Difficult to transfect all four plasmids into a single HEK 293T cell Fully differentiated neurons are not easily immortalized Specificity of transgene insertion into neuron genome cannot be controlled

26 Conclusions Preliminary culture techniques indicate a viable method for isolation and purification of kisspeptinexpressing cells in the AVPV and ARC AVPV and ARC explants adhere to tissue culture plate within four days of the initial harvest Neurons can survive in this environment for approximately six weeks before degrading

27 Conclusions Contamination easily preventable with penicillin, streptomycin, and gentamicin Post-sort, cells are stable in 20% FBS-containing DMEM with gentamicin for approximately four weeks Purified AVPV and Arc cultures are void of non-neuronal cell types

28 Conclusions Third generation lentiviral system appears to effectively confer immortalization on kisspeptin-expressing AVPV and Arc samples Proliferation begins approximately 4 weeks post-infection AVPV and Arc neurons retain unique expression pattern in response to estrogen

29 Cell Characterization + Do these cells retain their differential expression pattern? - Challenge efficacy RNA-sequencing to explore differential expression between neuronal populations Dynorphin A = Dyn Dyn Receptor = KOR Neurokinin B = NKB NKB Receptor = NK3R

30 Acknowledgements Patrick E. Chappell Ph.D Jadwiga M. Giebultowicz Ph.D Cheri Goodall Wanda Crannell College of Veterinary Medicine Grant