Oocyte–Targeted Deletion Reveals That Hsp90b1 Is Needed for the Completion of First Mitosis in Mouse Zygotes

Hsp90b1 is an endoplasmic reticulum (ER) chaperone (also named Grp94, ERp99, gp96,Targ2, Tra-1, Tra1, Hspc4) (MGI:98817) contributing with Hspa5 (also named Grp78, BIP) (MGI:95835) to protein folding in ER compartment. Besides its high protein expression in mouse oocytes, little is known about Hsp90b1 during the transition from oocyte-to-embryo. Because the constitutive knockout of Hsp90b1 is responsible for peri-implantation embryonic lethality, it was not yet known whether Hsp90b1 is a functionally important maternal factor.To circumvent embryonic lethality, we established an oocyte-specific conditional knockout line taking advantage of the more recently created floxed Hsp90b1 line (Hsp90b1(flox), MGI:3700023) in combination with the transgenic mouse line expressing the cre recombinase under the control of zona pellucida 3 (ZP3) promoter (Zp3-cre, MGI:2176187). Altered expression of Hsp90b1 in growing oocytes provoked a limited, albeit significant reduction of the zona pellucida thickness but no obvious anomalies in follicular growth, meiotic maturation or fertilization. Interestingly, mutant zygotes obtained from oocytes lacking Hsp90b1 were unable to reach the 2-cell stage. They exhibited either a G2/M block or, more frequently an abnormal mitotic spindle leading to developmental arrest. Despite the fact that Hspa5 displayed a similar profile of expression as Hsp90b1, we found that HSPA5 and HSP90B1 did not fully colocalize in zygotes suggesting distinct function for the two chaperones. Consequently, even if HSPA5 was overexpressed in Hsp90b1 mutant embryos, it did not compensate for HSP90B1 deficiency. Finally, further characterization of ER compartment and cytoskeleton revealed a defective organization of the cytoplasmic region surrounding the mutant zygotic spindle.Our findings demonstrate that the maternal contribution of Hsp90b1 is critical for the development of murine zygotes. All together our data indicate that Hsp90b1 is involved in unique and specific aspects of the first mitosis, which brings together the maternal and paternal genomes on a single spindle

The Human Proteins MBD5 and MBD6 Associate with Heterochromatin but They Do Not Bind Methylated DNA

BACKGROUND: MBD5 and MBD6 are two uncharacterized mammalian proteins that contain a putative Methyl-Binding Domain (MBD). In the proteins MBD1, MBD2, MBD4, and MeCP2, this domain allows the specific recognition of DNA containing methylated cytosine; as a consequence, the proteins serve as interpreters of DNA methylation, an essential epigenetic mark. It is unknown whether MBD5 or MBD6 also bind methylated DNA; this question has interest for basic research, but also practical consequences for human health, as MBD5 deletions are the likely cause of certain cases of mental retardation. PRINCIPAL FINDINGS: Here we report the first functional characterization of MBD5 and MBD6. We have observed that the proteins colocalize with heterochromatin in cultured cells, and that this localization requires the integrity of their MBD. However, heterochromatic localization is maintained in cells with severely decreased levels of DNA methylation. In vitro, neither MBD5 nor MBD6 binds any of the methylated sequences DNA that were tested. CONCLUSIONS: Our data suggest that MBD5 and MBD6 are unlikely to be methyl-binding proteins, yet they may contribute to the formation or function of heterochromatin. One isoform of MBD5 is highly expressed in oocytes, which suggests a possible role in epigenetic reprogramming after fertilization

Quand la mère est plus que responsable du devenir de sa progéniture : les mutations à effet maternel

Les mutations à effet maternel affectent les gènes qui codent pour des facteurs maternels (transcrits, protéines) normalement stockés dans l’ovocyte et utilisés par l’embryon après la fécondation. Alors que les femelles porteuses de ces mutations sont viables et d’apparence normale, le développement ou même la survie de leur descendance est compromis. Connues jusqu’ici chez les organismes inférieurs tels que la drosophile ou le poisson zèbre, les mutations à effet maternel (Dnmt, Hsf1 et Mater) ont été découvertes plus récemment chez les mammifères. Ces nouvelles données ouvrent la perspective d’une meilleure compréhension des mécanismes du développement embryonnaire qui sont directement sous le contrôle de l’ovocyte

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

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

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

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

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