Kume S et al. (1997), Developmental expression of the inositol 1,4,5-...

XB-ART-16886

Dev Biol 1997 Feb 15;1822:228-39. doi: 10.1006/dbio.1996.8479.

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Developmental expression of the inositol 1,4,5-trisphosphate receptor and structural changes in the endoplasmic reticulum during oogenesis and meiotic maturation of Xenopus laevis.

Kume S, Yamamoto A, Inoue T, Muto A, Okano H, Mikoshiba K.

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To study the development of the calcium release mechanism, we examined the temporal and spatial expression of inositol 1,4,5-trisphosphate receptor (IP3R) during the oogenesis and meiotic maturation of Xenopus laevis. Observation of a series of fixed samples by immunofluoresence microscopy revealed a relocalization of Xenopus IP3R (XIP3R)-positive structures during meiotic maturation. We visualized the endoplasmic reticulum (ER) using ER-sensitive dye, DiI, by observation under confocal laser scanning microscopy. Time-lapse visualization of the living oocytes revealed that while the ER of immature fully grown oocytes underwent relatively little movement, the ER of maturing oocytes and mature eggs moved rapidly. A possible role for the increase of ER mobility in the dynamic redistribution of XIP3R during oocyte maturation is also discussed.

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Species referenced: Xenopus laevis
Genes referenced: itpr1 mt-tr trna
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	FIG. 1. Expression of XlP3R during Xenopus oogenesis. (A) RNA profile. Total RNA equivalent to approximately four oocytes of stage 1- VI or In vitro mature eggs (lane +GVBD). and 20 l!g of yeast tRNA as a negative control, were analyzed by RNase protection assay for XlP3R transcripts and subjected to 8 M urea -5% PAGE. Arrowheads show the positions of the probe and the protected band. (B) Western blot analysis ustng antl-XlP3R polyclonal antibody (Kume et al.. 1993). Protein from a crude membrane fracuon equivalent to one oocyte was loaded onto 5% SDS- PAGE. Arrowhead shows the position of the XIP3R. The low-molecular-weight band (small arrow) Is a nonspecific band which appeared In U1e absence of anti-XIP3R when avldln - btotln complex methods were used for detection. (C) Densltomerlc analysts showing the relat ive Intens ity of the band corresponding to XIP3R. The lntenslUes or Xl P3R are plotted here as the fold Intensity relative to Immature fu lly grown stage Vl oocytes (the Intensity of immature fully grown stage VI = 1.0). Mean intensity and standard deviation are plotted. The number of experiments Is Indicated. The paired Student's t test was used to compare U1e values for each oocyte stage with stage Vl oocytes. The differences were significant for stage VII and rn oocytes and for mature eggs (marked with asterisks). with *P < 0.001 for stage 1m and rn oocytes; P < 0.05 for mature eggs.
	FIG. 2. Immunocytochemical characterization of XIP3R during oogenesis by light microscopy. Subcellular distributions of XIP3R at various stages of oocyte development detected by light microscopy using a XIP3R-specific polyclonal antibody. (A) XIP3R is distributed throughout the cytoplasm of stage I/II oocytes where yolk granules have not yet developed. (B) In early stage III oocytes, in which yolk granules have begun to develop, XIP3R is localized to streams of yolk-free cytoplasm throughout the oocyte surrounding the centrally placed GV. (C) In late stage III oocytes, which have begun to acquire polarity, XIP3R shows a distribution localized to the cortical region (white arrowheads) and perinuclear region (white arrow). (D) In late stage IV oocytes, the localization of XIP3R resembles that of fully grown stage VI oocytes. XIP3R is localized on the reticular network of intracellular structures (small arrow) and in the perinuclear region (large arrow). GV, germinal vesicle. Scale bar, 100 mm, A to D.
	FIG. 3. Immunohistochemical characterization of XIP3R meiotic maturation by light microscopy. (A) In immature fully grown stage VI oocytes, the perinuclear region as well as streams of yolk-free corridors are positively stained by anti-XIP3R antibody. Scale bar, 100 mm, A, C. (B) High magnification of the region within the white square in (A). XIP3R-positive streams of yolk-free corridors can be seen (white arrow). Scale bar 100 mm, B, D. (C) In maturing oocytes 6 hr after progesterone treatment at 187C (about 50% of oocytes underwent GVBD), large XIP3R-positive patches (large white arrow) are observed at the site where the GV was formerly located, and small XIP3Rpositive yolk-free patches are also observed in the cortical region (small arrow). (D) High magnification of the region with the white square in (C). XIP3R-positive yolk-free patches appear and streams of yolk-free corridors are no longer observable. GV, germinal vesicle.
	FIG. 4. (A) In meiotic mature eggs (100% GVBD; approximately 12 hr PG treatment at 187C), immediately after the white spot appeared in the animal pole indicative of GVBD, the large patches apparent in Fig. 3C (large white arrow) are no longer visible, and numerous small XIP3R-positive yolk-free patches are observable in both the deep cytoplasm (large white arrow) and the cortical region (small white arrow). Scale bar, 200 mm, A, C. (B) High magnification of the same mature egg in the region within the white square in (A). Numerous yolkfree patches have arisen in the cortical region. Scale bar, 100 mm, B and D. (C) The mature eggs were incubated for an additional 3 hr after 100% GVBD (15 hr PG treatment). Note that the XIP3R-positive cortical staining (white arrowhead) has become denser than that of (A). (D) High magnification of the mature egg in the region within the white square in (C). Cortical staining has increased. Compare the XIP3R-positive cortical staining indicated by white arrowheads in (D) with those in (B).
	FIG. 5. Quantitative analys is of the relative staining densi ty In the cortex versus the cytoplasmlc region 100 ~tm Interior to the cortex. The mean dens ities of fluorescence (subtracted with background dens ity) within regions A and Bare calculated. The numbers of oocytes or meiotically mature eggs are Indicated In parentheses. The regions for A. cortical staining. and B. Interior staining, are shown schematically In the lower panel. XlP3R gradually accumulates In the cortex with Increasing maturity. Bars show standard deviations. The paired Student's t test was used to compare the values for stage VI oocytes wtth mature eggs treated with progesterone for each time period. The differences are significant for mature eggs treated for 12 hr. •p < 0.05; and 15 hr, **P < 0.001.
	FIG. 6. An increase in ER movement during meiotic maturation. The ERs are stained by DiI injected into the cytoplasm. Images of ER are observed as membrane accumulations. Typical sequential images collected every 1 min (panels 1* to 4) from a single immature fully grown stage VI oocyte before (A) and after (B) meiotic maturation are shown. The optical images were taken at a 10-mm depth from the oocyte surface. Arrows of the same number show the same spots during the respective time sequences. Comparison of specific bright spots between time sequences reveals that: (A) ER in an immature stage VI oocyte undergoes little movement, whereas (B) ER in a meiotically mature egg moves rapidly. Note that in (B), brightly stained spot 1 moves left to right and then right to left during the time sequences from 1 min (1) to 4 min (4), spot 2 appears in the focal plane at 3 and then had moved rapidly away from the focal plane by 4, spots 3 and 4 change shape during the time sequences (1 to 4), spot 5 appears in 2 and has moved away from the focal plane by 3, while spot 6 moves away from the focal plane at 2 and has reappeared by 3. Spot 7 stained most brightly in 1 and has dimmed somewhat by 2, remains stationery at 2 and 3, and has dimmed again by 4 and the following image (data not shown). Scale bar, 4 mm. (C) The images are further processed by extraction followed by thresholding the edges in order to obtain the outline of each image. An overlay of the traces of the ER at four sequential time points is shown to demonstrate the movement of particular ER accumulations shown in (A), upper panel, and (B), lower panel, respectively. In immature fully grown oocytes, the traces shown in different colors nearly overlap and result in numerous black lines, whereas in meiotically mature eggs, the ER moves so rapidly that the traces do not overlap and thus the individual colors of each trace are observed. 1* in red, 2* in blue, 3* in green, and 4* in pink, for both the upper and the lower panels.
	FIG. 7. Histograms of the velocity (distance moved per minute) of each ER accumulation in immature fully grown stage VI oocytes (open columns) and meiotically mature eggs (closed columns). Bars represent standard deviations. Data collected from three representative sets of oocytes and meiotically mature eggs are used in the quantitative analysis. The distribution of the velocities of ER accumulations in mature eggs showed a significant shift to a higher velocity compared to that of oocytes (Kolmogorov-Smirnov (K-S) test, P 0.001).