Sperm contents exclude maternal yolk granules and mitochondria but not ER.

(A) Time-lapse in utero images of control embryo expressing GFP::SPCS-1 (signal peptidase) and mKate::TBA-2 (tubulin). ER morphology transitions from sheet-like during metaphase I and metaphase II to dispersed during anaphase I and anaphase II. (B) Image of a fixed metaphase I embryo expressing GFP::VIT-2 (maternal yolk granules), paternal mitochondria labelled with MitoTracker Deep Red FM, and stained with alpha tubulin antibody and DAPI. (C) Image of fixed late metaphase I embryo expressing GFP::SPCS-1 (maternal ER), paternal mitochondria labelled with MitoTracker Deep Red FM, and stained with alpha tubulin antibody and DAPI. (D) Image of a fixed metaphase I embryo expressing COX-4::GFP (maternal mitochondria), paternal mitochondria labelled with MitoTracker Deep Red FM, and stained with alpha tubulin antibody and DAPI. (E) Time-lapse in utero images of an embryo expressing HALO::ER (HALO tag with signal peptide and ER retention signal from HSP-3/BiP), VIT-2::GFP, paternal mitochondria labelled with SDHC-1::mCherry (succinate dehydrogenase). Images demonstrate sperm contents streaming in the short axis of the embryo. (A-E) Bars: (whole embryo) 10 um; (inset) 2 um.

Maternal mitochondria are still excluded from the sperm contents in kca-1(RNAi) embryos.

(A) Images of fixed meiotic embryos expressing COX-4::GFP (maternal mitochondria) that have been mated with MitoTracker Deep Red FM stained fog-2(q71) males. In the control, maternal mitochondria are packed inward away from the cortex (n=11). Paternal mitochondria take up a volume excluding maternal mitochondria. In kca-1(RNAi) embryos, maternal mitochondria extend to the plasma membrane but paternal mitochondria still occupy a volume excluding maternal mitochondria (n=8). (B) Image of fixed Caenorhabditis macrosperma meiotic embryo mated with MitoTracker Deep Red FM stained males. The paternal mitochondria take up a larger volume than in C. elegans but the cohesion of mitochondria near paternal DNA is still conserved between species (n=4). (A-B) Bars: (whole embryo) 10 um; (inset) 5 um. White dotted boxes denote area of insets.

Maternal ER enters the sperm contents after a delay.

(A and B) Two different time-lapse sequences of fertilization of oocytes expressing TMCO-1::GFP::SSPB (maternal ER) and mCherry::PH (maternal plasma membrane) by sperm labelled with MitoTracker Deep Red FM (paternal mitochondria). (A) Sperm-egg fusion occurs at 0:00. Maternal ER ER invades the sperm cytoplasm at 1:25. (B) The envelope of maternal ER around the sperm DNA is first visible at 7:24. Bars: (whole embryo) 10 um; (inset) 5 um. White dotted boxes denote area of insets.

Maternal BAF-1 associates with sperm DNA during anaphase I indicating that the ER envelope is not sealed.

(A) Time-lapse in utero images of an embryo expressing mCherry::histone and GFP::BAF-1. Association of BAF-1 during anaphase indicates that maternal BAF-1 associates after assembly of the ER envelope. Bar, 5 um. (B) Fixed embryos during metaphase I and anaphase I. Maternal GFP::BAF-1 strongly localizes to both maternal and paternal chromosomes during anaphase I, but not metaphase I. Bars: (whole embryo) 10 um; (inset) 2 um. (C) Fluorescence intensity of GFP::BAF-1 on maternal and paternal chromosomes. During anaphase I, there is an increase in GFP::BAF-1 on both the maternal and paternal chromosomes. ** p<0.01, *** p<0.001 by Mann-Whitney U Test.

MEI-1katanin and KLP-7kinesin-13 limit meiotic cytoplasmic streaming of the sperm contents.

(A) Illustration of the types of movement that can be quantified from tracks of sperm contents. (B) Example time-lapse sequences showing movement of the maternal ER envelope around the sperm DNA and paternal mitochondria during meiosis. The bottom row shows MTrackJ tracks of the ER envelope around the sperm DNA over the length of meiosis. Cell cycle stage was determined from the morphology of the ER. Minimal displacement was observed during metaphase I (MI, red track). Most displacement was observed during anaphase I (AI, yellow track). In klp-7(RNAi), movement continued through metaphase II (MII, green track) which was prolonged in klp-7(RNAi) embryos. Bar, 10 um. (C) Maximum 2D long axis displacement and maximum short axis 2D displacement during individual cell cycle stages. * p< 0.05, ** p< 0.01, ** p< 0.001 by Mann-Whitney U Test.

ATX-2 is depleted by 3 different methods.

(A-C) Single plane images of living -1 oocytes. (A) Background autofluorescence of N2 (wild-type) with no GFP. Drawn dotted lines show the outline of oocytes and germinal vesicles (nuclei). (B-C) Live images of -1 oocytes in strain with endogenous ATX-2::AID::GFP treated with no auxin, 1 hr auxin, 27 hr control L4440(RNAi), 27 hr atx-2(RNAi) or 24 hr gfp(RNAi). Bars, 10 um. (D) Mean GFP fluorescence in the cytoplasm of -1 oocytes after each treatment. **** p< 0.0001 by Welch’s t-test and Brown-Forsythe test.

Paternal mitochondria scatter during meiosis after ATX-2 depletion.

(A-E) Maximum intensity projections of z-stacks of fixed meiotic embryos stained with tubulin antibodies and DAPI and with paternal mitochondria labeled by mating with MitoTracker Deep Red FM treated fog-2(q71) males. (A) N2 wild-type embryos treated with control L4440(RNAi) or atx-2(RNAi). (B, C, E) Embryos expressing TIR1, GFP::SPCS-1, mKate::TBA-2, and with endogenously tagged ATX-2::AID::GFP. (B) 27 hr control L4440(RNAi) or atx-2(RNAi). Arrows denote mitochondrial fluorescence from sperm outside the embryo overlapping with the embryo as a result of the maximum intensity projection. (C) No auxin or 1 hr auxin treatment. (D) GFP(RNAi) on strain with no tag on ATX-2 but expressing GFP::SPCS-1 and mKate::TBA-2. GFP::SPCS-1 fluorescence remains because SPCS-1 and ATX-2 are tagged with GFPs with different sequences. (E) Control L4440(RNAi) or gfp(RNAi) of ATX-2::AID::GFP strain. Bars, 10 um. White dotted outlines indicate the cortex of the cell.

Quantification of paternal mitochondrial scatter in ATX-2-depleted anaphase I meiotic embryos.

(A) Mean and (B) standard deviation of the distance of individual paternal mitochondria from the sperm DNA determined from Z-stacks of fixed anaphase I embryos. Each dot represents one embryo. Distances for individual mitochondria are in Supplementary data file.

Capture of the sperm DNA by the meiotic spindle in KLP-7 ATX-2 double depleted meiotic embryos.

(A) Low magnification and (B) high magnification time-lapse images of 47 hr klp-7(RNAi) + 1 hr auxin meiotic embryo expressing GFP::SPCS-1 (ER), mKate::TBA-2 (Tubulin), ATX-2::AID::GFP and with paternal mitochondria labeled by mating MitoTracker Deep Red FM treated fog-2(q71) males. (C) Time-lapse images of a KLP-7 ATX-2 double depleted embryo in which the maternal ER envelope around the sperm DNA is transiently captured and stretched in the direction of cytoplasmic streaming. Bars: (whole embryo) 10 um; (inset) 5 um.

Distance of the sperm contents from the cortex of control embryos at metaphase I vs anaphase I.

Figure S2: Localization of ATX-2 in -1 oocyte and +1 meiotic embryo.

Deconvolved single plane images from z-stacks acquired on a spinning-disk confocal. A. -1 oocyte (n=10). B. Metaphase meiotic embryo (n=10). C. Anaphase meiotic embryo (n=4). ER labeled with HALO-tag with the signal peptide and ER retention signal from HSP-3. Endogenous ATX-2::AID::GFP. mKate::Tubulin. Paternal mitochondria labeled with SDHC-1::mCherry. All Bars= 10um.

Cytoplasmic streaming after ATX-2 depletion.

Maximum displacement of the sperm contents during any meiotic cell-cycle phase in ATX-2::AID::GFP embryos with or without 1 hr auxin.