HSPA5 expression but not localization is modified in MT embryos.

<p>(<b>A</b>) RTqPCR was used to determine the level of Hspa5 transcripts in WT and MT samples: GV oocyte, ovulated MII oocytes, 1-cell or zygote (30 hours post hCG) and 2-cell (42–44 hours post hCG) embryos. Relative quantities of mRNA were normalized against the quantity of ribosomal S16 transcripts and Hspa5 transcript level in GV oocytes was arbitrarily given the value 1. (<b>B</b>) HSPA5 was immunodetected by Western blot using WT and MT samples as indicated. Experimental procedure was similar as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017109#pone-0017109-g002" target="_blank">Figure 2C</a>. The number written below each lane corresponds to the relative densitometric value (HSPA5/alphaTUB.). (<b>C</b>) WT embryos (pronuclear -PN5- and mitotic 1-cell stages) and MT mitotic zygotes were prepared for immunofluorescence as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017109#s4" target="_blank">materials and methods</a>. Representative images show that ER chaperones HSPA5 and HSP90B1 did not completely colocalize. HSP90B1 was found within the spindle structure but not HSPA5 as shown in inset (i). HSPA5 and HSP90B1 were both present in the granular region surrounding the spindle but higher magnification (inset ii) showed that even in this region where both ER chaperones were present, they were not fully colocalized. HSPA5 seemed to be more abundant in the cortex region of the zygote. HSPA5 staining was not visibly modified in MT embryos.</p

Development of embryos obtained by crossing <i>Zp3-cre; Hsp90b1^flox/flox</i> (MT) or control females with wild-type (WT) males.

<p>Development of embryos obtained by crossing <i>Zp3-cre; Hsp90b1^flox/flox</i> (MT) or control females with wild-type (WT) males.</p

Peri-spindle accumulation of filamentous actin in mutant embryos.

<p>(<b>A</b>) Representative pictures of WT and MT zygotes at the metaphase stage of the first mitosis stained with phalloidin (green: filamentous actin) and TO-PRO-3 (blue: DNA) shown with original contrast and enhanced contrast to better visualize actin accumulated around the spindle. (<b>B</b>) Representative examples of plot profiles (Image J) analyzing the actin staining (normal contrast) along the diameter section of the zygotes. (<b>C</b>) Graph showing the relative intensity of actin staining measured at the peri-spindle region and cortex in WT zygotes (n = 5) and MT zygotes (n = 13). Data presented as mean value (A.U.: arbitrary unit). p<0.01.</p

G2/M block and abnormal mitosis in mutant embryos.

<p>(<b>A</b>) The size of both maternal (expected to be smaller) and paternal (expected to be larger) pronuclei was measured in one–cell WT and MT embryos at 29–30 hours post hCG in order to determine their progression in the G2 phase of the first cell cycle. No significant difference was found between WT and MT pronuclei. (<b>B</b>) One-cell mutant embryos were analyzed at 42–44 hours post hCG to determine the percentage of embryos blocked at the G2/M transition or during mitosis. (<b>C</b>) One-cell mutant embryos were analyzed at 34 and 42–44 hours post-hCG and classified according to their nuclear or spindle morphology. NB: WT embryos had mostly reached the 2-cell stage at 34 hours posthCG (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017109#pone-0017109-g005" target="_blank">Figure 5</a>). (<b>D</b>) Tubulin (green) and DNA (blue) fluorescent staining of WT and MT embryos. Representative images of mitotic (i) and 2-cell (ii) WT embryos. Spindle morphologies of MT embryos are illustrated from iii to vi: (iii) two groups of chromosomes; (iv) multipolar spindle; (v) central organization of the spindle with peripherical chromosomes; (vi) a ‘normal’ bipolar spindle. Insets show the corresponding zygote.</p

Thinner zona pellucida in HSP90B1 deficient oocytes.

<p>(<b>A</b>) Representative images of wild-type (WT) and mutant (MT, obtained from Zp3-cre<i>; Hsp90b1^flox/flox</i> females) ovulated oocytes showing that zona pellucida is thinner in mutant oocytes. (<b>B</b>) Ratio between thickness of zona pellucida and oocyte diameter was calculated and indicated in arbitrary unit (A.U.). Mutant zona pellucida (n = 99) were significantly thinner than wild type ones (n = 36). p<0.001. (<b>C</b>) Histological sections of adult ovaries were prepared for immunofluorescence against ZP2 and representative examples of WT and MT oocytes from secondary follicles are shown. Arrows indicate the ZP2 positive structures in mutant oocytes.(<b>D</b>) Percentage of WT and MT oocytes containing ZP2 positive structures at late primary, secondary and tertiary follicular stages. Numbers indicated above the bar correspond to the number of follicles for each category.</p

LCA-FITC staining of membranous organelles (ER-nucleus, Golgi apparatus).

<p>Representative pictures of WT and MT zygotes at the metaphase stage of the first mitosis stained with LCA-FITC (lens culinaris agglutinin-fluorescein complex). Chromosomes were stained with TO-PRO-3. WT zygotes exhibited a granular LCA positive region surrounding the spindle which was not found in some MT zygotes (shown in upper row). Other MT zygotes displayed some LCA positive granules within the structure of the spindle (shown in lower row, arrowhead).</p

HSP90B1 is not essential for meiotic maturation.

<p>(<b>A</b>) Mean percentage of GV (WT and MT) oocytes which reached MII stage after in vitro culture. (<b>B</b>) MII oocytes were processed for microtubule, actin and DNA staining. Arrowheads indicate the MII chromosomes and arrows show the first polar body resulting from the first meiotic division (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017109#pone-0017109-g001" target="_blank">Figure 1A</a>). (<b>C</b>) Chromosome spreads were prepared using MII (WT and MT) oocytes. They show chromosomes with 2 chromatids as expected before the 2^nd meiotic division.</p

Ovary histology and Hsp90b1 expression.

<p>(<b>A</b>) Histological sections of wild-type (WT) and Zp3-cre<i>; Hsp90b1^flox/flox</i> (MT) ovary were HE stained WT and MT sections contain follicles at different stages including visible oocytes (arrow head) and corpus luteus (arrow). Scale bar: 0.5 mm. (<b>B</b>) RTqPCR was used to determine the level of Hsp90b1 transcripts in WT and MT fully grown oocytes (GV stage). Relative quantities of mRNA were normalized against the quantity of ribosomal S16 transcripts and Hsp90b1 transcript level in WT oocytes was arbitrarily given the value 1. (<b>C</b>) Western blot analysis of HSP90B1 protein content in fully grown (GV), metaphase II (MII) oocytes and in 1-cell, 2-cell embryos. Samples collected from WT and MT females are shown as indicated. Membranes were reprobed with anti alpha-tubulin (as aTUB.) as loading control. (<b>D</b>) HSP90B1 immunodetection was performed on ovary histological sections. Left panels (WT) show HSP90B1 expression in both somatic cells and oocytes at various follicular stages. Middle panels (MT) illustrate the oocyte specific loss of HSP90B1 in MT sample. Right panels (MT) show 3 primary follicles containing an oocyte section. HSP90B1 is strongly immunodetected in granulosa cells while it is barely detectable in oocyte cytoplasm. Arrowheads indicate GV oocytes included in the sections.</p