2. Methods 2010 - Techniques in Reproductive Biology...

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Techniques in Reproductive Biology
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Bioassay Use of a known biological response Develop a dose response curve Access unknowns Still extensively used Estradiol Estradiol 1ppt 1ppt 100ppt 100ppt 1ppb 1ppb 100ppb 100ppb 10ppm 10ppm t 0 0 20 20 40 40 60 60 80 80 100 100 Sex Reversal % Female @ 33°C
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Alligator Sex Reversal t-Nona t-Nona DDD DDD DDD+tN DDD+tN Dicofol Dicofol Estradiol Estradiol 0 0 20 20 40 40 60 60 80 80 100 100 1ppt 1ppt 100ppt 100ppt 1ppb 1ppb 100ppb 100ppb 10ppm 10ppm All compounds estrogenic - but not ‘complete estrogen’
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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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CPM 1 CPM 2 MEAN CPM CPM 1 CPM2 TC 14595 15187 14891 NSB 87 96 92 B0 0 2919 2898 2909 STD 1 1.5625 2862 2868 2865 STD 2 3.125 2750 2719 2735 STD 3 6.25 2614 2672 2643 STD 4 12.5 2352 2434 2393 STD 5 25 2014 1980 1997 STD 6 50 1414 1388 1401 STD 7 100 935 869 902 STD 8 200 535 511 523 STD 9 400 308 339 324 STD 10 800 209 206 208 Testosterone RIA -500 0 500 1000 1500 2000 2500 3000 3500 4000 1 10 100 1000 10000 T (pg/ml) C P M Series1 Log. (Series1) •1900 cpm = 28 pg/tube •If: •100 μ l/tube •Then •Concentration in ml •10 X 28 pg/tube •280 pg/ml
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Guillette et al. (1994) Environ. Health Perspec. 102:680-688 Plasma Estradiol (pg/ml) Juvenile Alligators - 9 mo old Juvenile Alligators - 9 mo old 0 20 40 60 80 100 120 140 Male Female Apopka Woodruff ! Plasma Testosterone (pg/ml) Juvenile Alligators - 9 mo old Juvenile Alligators - 9 mo old 0 10 20 30 40 50 60 Male Female Apopka Woodruff *
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Surgery Earliest technique 1) “Extirpation” Remove tissue 2) See what happens 3) Replacement Still used extensively Interpretation can be difficult
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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
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X-ray of gravid tuatara eggs eggs too
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MRI of an Alligator egg Hind limb Heart Eye Water in allantois
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