Techniques in Reproductive Biology

Transcription

1 Techniques in Reproductive Biology

2 Bioassay Sex Reversal % 33 C ppt Use of a known biological response 80 Develop a dose 60 response curve Access unknowns 40 Still extensively used ppt 1ppb 100ppb 10ppm 0 t-nona DDD 0 DDD+tN Dicofol Estradiol t-nona DDD DDD+tN Dico

3 Radioimmunoassay Used to measure quantitatively hormone concentrations in blood, receptors in tissue, etc. A competitive binding assay using Radio = radioactive label Immuno = specific antibody Assay = quantitative approach

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5 CPM 1 CPM 2 MEAN CPM CPM 1 CPM2 TC NSB B STD STD STD STD STD STD STD STD STD STD Testosterone RIA 1900 cpm = 28 pg/tube If: 100 µl/tube Then Concentration in ml 10 X 28 pg/tube 280 pg/ml C P M Series1 Log. (Series1) T (pg/ml)

6 Plasma Estradiol (pg/ml) Juvenile Alligators - 9 mo old ! Apopka Woodruff Plasma Testosterone (pg/ml) Juvenile Alligators - 9 mo old 0 Male Female * Apopka Woodruff Guillette et al. (1994) Environ. Health Perspec. 102: Male Female

7 Surgery Earliest technique 1) Extirpation Remove tissue 2) See what happens 3) Replacement Still used extensively Interpretation can be difficult

8 X-Ray & Other Imaging Modern imaging technology extensive X-ray; MRI; CAT scan Used for non- to minimally invasive view Can provide extensive information Laparoscopy of Tuatara ovary

9 X-ray of gravid tuatara eggs too eggs

10 MRI of an Alligator egg Hind limb Heart Eye Water in allantois

11 Histology Sections of tissues cut at micrometer thickness or smaller Provides view of tissue at cellular or subcellular level Initial process of many other processes

12 SEM/TEM Electron microscopy allows sub cellular view of cells Powerful tools for anatomy and physiology SEM: tissue dried - coated with gold TEM: tissue fixed - cut at < 1 µm

13 SEM of uterine surface with Egg shell fibers extruding

14 Histochemistry Used to examine specific enzymatic reactions of cells Can be used to identify pathological tissues Or live and dead tissue Bull sperm live - green dead - red

15 Immunocytochemistry Used to identify location of protein in a cell/tissue Use of a specific antibody and targeted enzyme reaction

16 Aromatase Testis tubules Immunocytochemistry Product Enzyme + Avidin Secondary antibody + Biotin Slide Tissue Primary antibody Protein Relaxin -Corpus luteum

17 ICC for ER in Uterine tissue

18 In situ Hybridization Used to localize specific mrna in tissue or cell Targeted probe complementary to the mrna

19 Electrophoresis Use molecular weight &/or charge to separate chemicals proteins or RNA or DNA commonly isolated using electrophoresis 1 or 2 dimensions

20 2D-SDS PAGE for Oviduct proteins in gator 1D-SDS PAGE for Serum proteins in fish

21 Western blots Protein from electrophoretic gel transferred to membrane Stained with a specific antibody ICC on gel blot Identifies a specific band associated with a specific protein

22 1D PAGE for Vtg Western for Vtg

23 RNA/Northern Blot Electrophoresis of RNA Transfer to membrane and probe with complementary radioactive cdna Expose X-ray film/plate

24 FEMALE MALE Ovary Uterus Oviduct Mammary -RT Pituitary Hypothalamus Olfactory Bulb Cortex Heart Aorta Liver Kidney Lung Sk. Muscle Testes Prostate Epididymis ERα ERβ β-actin JF Couse and KS Korach (1999) Endocrine Reviews. 20: JF Couse et al. (1997) Endocrinology. 138: Couse, 1999

25 DNA Sequencing

26 H 3C OH HO H 3C OH HO H 3C OH ER HO HO H 3C OH ER HO H 3C OH ER OH H 3C ER HO RNA Pol ER ERE TATA ER

27 Phylogeny of ER Katsu, et al., Gen. Comp. Endo. (2003)

28 Transfected Cell Line Develop a test organism Insert a receptor-gene construct if interest for testing Human ER or AR with reporter gene

29 Reporter Gene Assay using ERE-Luc Test Chemicals Receptor expression plasmid ERα Luminescence ERE Firefly Luciferase cdna Transcription Reporter plasmid Translation Reporter protein

30 Yeast Estrogen Screen (YES) Assay Miller Units - β-galactosidase activity 30 E 2 25 DES o,p'-ddt OP concentration (nm) Arnold et al., 1996, Environ. Health Perspec.

31 PCR Use of natural DNA/RNA mechanisms