Fig. S1. Identification of novel YP proteins. (A) The YP purification and fractionation scheme. (B) Coomassie-stained SDS-PAGE gels of purified YPs and derived fractions, the YP supernatant and YP membranes.

Fig. S2. A novel protein cross-reacts with the anti-Seryp antibody. Early stage oocytes, and occasionally later stage oocytes, contain a novel protein (p60) that strongly cross-reacts with the anti-Seryp antibody (α-Seryp). p60 could represent a fragment of Seryp covalently bound to the active site of a target protease present in oocytes. Oocytes at different stages were collected from a different female than in Fig. 2 in the main text and western blotted in three developmental series. Protein concentration in oocyte extracts was approximately normalized by oocyte volume prior to loading equal volumes of extract onto the gel. In total, of oocyte stages I-III, 8 of 9 contained abundant p60 and 5 of 9 contained abundant p50. Of oocyte stages IV-VI, 2 of 11 contained abundant p60 and 11 of 11 contained abundant p50.

Fig. S3. SERYP transcripts are detectable in maternal liver but not in oocytes, eggs or embryos. qRT-PCR demonstrated that there is not sufficient SERYP transcript present in oocytes to account for the ∼0.9 g of protein that accumulates during oogenesis. cDNA was prepared from total RNA isolated from all six oocyte stages, unfertilized eggs, and four early embryonic stages. The amount of β-ACTIN (gray bars), ODC (black bars) and SERYP (open bars) transcript present in each experimental RNA sample was quantified using qRT-PCR Taqman assays. Transcript quantity is reported as the amount of positive control total RNA (isolated from the liver of a β-estradiol-induced female) that contains an equivalent amount of transcript. Stage VI oocytes, eggs and early embryos contain ∼5 g of total RNA (Gurdon and Wakefield, 1986). None of the experimental samples had detectable SERYP transcript. But the equivalent of 100,000 in vitro-transcribed SERYP transcripts spiked into total RNA from a stage 26 embryo could be detected by this assay. This detection limit amounted to 2500 in vitro SERYP transcript equivalents per qRT-PCR well, or ∼100,000 transcripts per oocyte, egg or early embryo. This sensitivity corresponds to detecting approximately one transcript per cell in tissue culture cells, which are ∼5 pL compared to the ∼0.5 L of cytoplasm present in full-grown oocytes (Gurdon and Wakefield, 1986). Even assuming (1) the most generous estimates of translation rate per ribosome from bacteria (∼40 amino acids/second, ∼40× as fast as the published X. laevis rate) (Gurdon and Wakefield, 1986), (2) maximal ribosome density per SERYP transcript (100 bp/ribosome) (Gurdon and Wakefield, 1986), and (3) maximum time of oocyte development (8 months) (Zhou et al., 2004), it is not possible for less than 100,000 transcripts to generate the ∼0.9 g of Seryp protein present in full-grown oocytes and eggs. Measurements are the average of three biological replicates, each measured in triplicate by qRT-PCR. Error bars extend one s.e. in either direction. Control reactions lacking reverse transcription were conducted for every sample to confirm that all measurements were of cDNA, not genomic DNA or other contaminants (data not shown). cDNA prepared from the indicated amounts of liver total RNA (200 pg-2.5 g) was used to construct standard curves relating liver total RNA to threshold cycle (C_T). The C_Ts from the liver samples were then referenced back to these standard curves and the derived total RNA plotted below the experimental samples to demonstrate internal consistency.

Zhou, Y., Zhang, J. and King, M. L. (2004). Polarized distribution of mRNAs encoding a putative LDL receptor adaptor protein, xARH (autosomal recessive hypercholesterolemia) in Xenopus oocytes. Mech. Dev. 121, 1249-1258.

Fig. S4. YP consumption is an intracellular process in the developing gut. In an immunostained (anti-Vtg-Alexa488, anti-Seryp-Alexa568) transverse section, the intestinal loops of a tadpole stage embryo (stage 43) were filled with activated (i.e. Seryp⁻ Vtg⁺) YPs. Sections were imaged at low magnification (top) and select regions (yellow rectangles) were imaged by confocal microscopy (bottom). Scale bars: 100 m (top); 20 m (bottom). In confocal micrographs from the gut, developing intestinal loops with and without Seryp could be seen in the same animal.

Fig. S5. Non-yolk proteins accumulate during development. Extracts were prepared from an individual egg (e) or from individual embryos at the indicated hours of development post-fertilization (pf) and Nieuwkoop-Faber (N-F) stage. One out of 50 individual eggs or embryo extracts was separated by SDS-PAGE and the gel was stained with Coomassie. During embryogenesis, the major yolk proteins LV1 and LV2 disappear (brackets), whereas numerous highly abundant proteins accumulate (arrows).

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