29
30
31
32
25
26
27
28
21
22
23
24
17
18
19
20
13
14
15
16
9
10
11
12
5
6
7
8
1
2
3
4
41
42
43
44
45
46
37
38
39
40
33
34
35
36
Electronic Supplemental Materials
Materials and Methods
2.1 Egg collection, treatment and incubation
Infertile eggs were taken from a pen containing only females, but housed such that they were exposed to males. Regardless of treatment group, eggs were incubated at 37.8°C with 64-68% humidity in a 1502 Sportsman Incubator (GQF Manufacturing Co.). After their respective periods of incubation, eggs were removed from the incubator, wrapped in tin foil, and placed into a -20°C freezer. All experiments on egg components were done within 11 months of [
3
H]-corticosterone injection. Given that the half-life of tritium is
12.3 years we did not lose a significant amount of tritium activity to radioactive decay during that time. In addition, all data were analyzed in terms of proportions and percent total radioactivity recovered, rather than raw radioactivity levels.
2.3 Thin Layer Chromatography
The remaining portions of homogenized yolk and embryo samples were extracted with methanol in a 1:5 (g:mL) ratio. Samples were then centrifuged at 950 x g for 10 minutes at -4°C using an Avanti J-E centrifuge (Beckman Coulter). Aliquots of the supernatant were diluted in a 1:9 ratio (v:v) with distilled water and passed through Sep-Pak C18 columns (Waters Inc.) preconditioned with 5mL of methanol and 5mL of distilled water.
Ether-soluble steroids were eluted from the column using 5mL of ethyl ether while watersoluble steroids were eluted using 5mL of methanol. The ether-soluble and water-soluble portions were dried under a stream of nitrogen gas and resuspended in 100μL of ethanol.
A 20μL aliquot of each portion was counted in a scintillation counter to determine the ratio of ether-soluble to water-soluble corticosterone for each developmental time point.
Ether-soluble samples from ID12 and beyond did not possess enough radioactivity to run
TLC and so they were excluded. Sample was spotted onto aluminum backed silica TLC plates with a 254nm fluorescence indicator (Sigma-Aldrich). An internal standard composed of androstenedione, testosterone (Sigma-Aldrich), cortisol, corticosterone, and dihydrocorticosterone (Steraloids) was spotted with each sample. Identical plates of each sample were developed in two solvent systems. The first was 95:5 chloroform:methanol
(v:v) which allowed us to separate free steroids from one another to determine if corticosterone was being converted into another free steroid. The second was 5:5:1 ethanol:ethyl ether: ammonium hydroxide (v:v:v) to separate free (water-soluble) steroids, from conjugated (ether-soluble) steroids (in particular the two major classes of steroid conjugates glucuronides and sulfates) After each plate was developed once, the movement of radioactivity on the plate was characterized by cutting lanes into 15 0.5 cm x 1.0cm sections and counting each section [5]. Plates were standardized by multiplying individual sections by the average ratio of free:conjugated corticosterone found at that developmental time point.
Results
Supplemental Figure 1: TLC allows for the separation of similar chemical species on a
TLC plate based on differential migration rates. Plates were separated into 15 sections

51
52
53
54
55
47
48
49
50 with plate section 1 representing the point of greatest migration (least polar) and plate section 15 the point of least migration (most polar). Samples were spotted onto section
15. Representative standardized TLC plates from ID9, ID12, and ID15 confirm the accumulation of metabolized corticosterone in the embryo. (a) The 95:5 solvent system separates free and conjugated steroids. Standards migrated according to polarity
(A
4
=Androstenedione, T=Testosterone, B=Corticosterone, F=Cortisol,
DHB=Dihydrocorticosterone). (b) The 5:5:1 solvent system separates glucuronide conjugates from sulfate conjugates. Brackets indicate the migration pattern for free steroids, sulphate conjugates, or glucuronide conjugates.