TIME-COURSE OF THE TYPE I InsP^R DECREASE AFTER FERTILISATION

3.7.1 The lev el o f type I InsPjR decreases w ithin eight hours o f fertilisation

I investigated in more detail the downregulation of type I InsP^R protein that I previously showed to take place by the late 2-cell stage. In order to determine at which point in development during progression to the 2-cell stage this decrease takes place, I initially carried out Western blotting on pronucleate embryos frozen for analysis 8 hours after exposure to sperm. 180 pronucleate embryos were subjected to Western analysis parallel to controls of MU oocytes and age-matched MR oocytes which had been incubated alone in T6 culture medium (Fig. 3.7.A). Analysis of type I InsP^R levels indicated that the level of protein was similar in pronucleate and late 2-cell embryos, suggesting that downregulation was complete 8 hours after addition of sperm (Fig, 3 .7 .B ). Similar results were obtained using two type I-specific antibodies, Ab40 (n=4) and CTl (n=3). This decrease in type I InsP^R protein was specific to fertilised embryos as unfertilised oocytes incubated (aged) in T6 medium for the same amount of time (8 h) showed roughly similar levels of protein as the freshly ovulated oocytes (Fig. 3.7.A and B). Collating results from between 6 and 14 Western analyses for aged eggs or fertilised embryos showed that the only significant change in InsP^R levels occurred between MR stage oocytes and pronucleate embryos (8 hours after exposure to sperm) (Fig. 3.7.C).

3.7.2 Im m u n olocalisation of type I InsP^R before and eight hours after fertilisation

To examine whether there was any spatial regulation in the loss of type I InsP^R after fertilisation, I localised type I using the previously characterised CTl antibody and confocal microscopy (Fig. 3.8). After staining with C Tl, immunoreactive protein was distributed throughout the MR oocyte cytoplasm (Fig. 3.8.A). The meiotic spindle (section through spindle not shown) was devoid of any staining in all 12 of the mature oocytes examined. Individual bright spots, possible non-specific clustering of antibodies, overlay a network­ like pattern of staining which may reflect the presence of type I InsP^R distributed throughout the endoplasmic reticulum. No staining was detectable in the absence of the antibody, suggesting that the immunoreactivity was specific (Fig. 3 .8 .C ). Furthermore, the staining was neutralised by the peptide antigen used to raise the antibody (not shown). Staining with CT2 antibody did not label the mature oocytes, consistent with my Western analysis result.

% :

Mil Aged Pn

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Mil Aged Pn Late 2-cell

C 125 n 100 - ' ô g. 75 -

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Mil Aged Pn Late 2-cell

Figure 3.7 The level of type I InsP^R decreases within eight hours of fertilisation. Western analysis of InsP^Rs in fertilised pronucleate (Pn) embryos collected 8 hours after exposure to sperm, and late 2-cell embryos.

Protein from 180 Mil oocytes, age-matched Mil oocytes (Aged), pronucleate (Pn) embryos or 2-cell embryos was subjected to electrophoresis and immunoblotted with the type I-specific antibody CTL (A) Pn stage embryos show a decrease in type I InsPgR levels but age-matched unfertilised control oocytes show similar levels of InsPgR as freshly ovulated Mil oocytes. (B) The decrease in type I levels by Pn stage is as extensive as that seen by the late 2- cell stage. The data in (A) and (B) is representative of at least 3 experiments for each stage of development, using both Ab40 and CTl antibodies. (C) Optical densities of immunoreactive type I bands from up to 14 Western analyses for aged eggs or fertilised embryos were calculated using NIH image as described in section 3.5. Bars with common superscripts are not

Figure 3.8 Immunolocalisation of type I InsPjR before and after fertilisation. The distribution of type I InsPgR was examined in ovulated Mil oocytes and pronucleate (Pn) embryos fixed 8 hours after fertilisation, using the type I-specific antibody CTl and confocal microscopy. (A) Subcortical and cortical sections are shown. Immunoreactive protein is distributed throughout the Mil oocyte cytoplasm. The approximate position of the spindle (outside section) is indicated. (B) Immunoreactive protein remains dispersed throughout the fertilised Pn embryo cytoplasm and the second polar body but is absent from the pronuclei. In the absence of primary antibody (C) or after preadsorption with the peptide antigen (not shown), there was no detectable staining. (D) Mean pixel intensities taken from projected images of 5 Mil and fertilised oocytes reveals a significant difference in staining intensity (t-test: P<0.01).

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Metaphase II Metaphase II (cortex)

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Fertilised Pn stage Fertilised Pn stage (cortex)

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In fertilised pronucleate embryos (fixed 8 hours after exposure to sperm) the pattern of immunofluorescent staining remained dispersed throughout the cytoplasm and, as expected, was not present within the pronuclei (Fig. 3 .8 .B ). After identical processing and at the same confocal settings it was observed that the staining intensity was consistently (9/9) decreased in fertihsed embryos (8 hours after fertilisation), consistent with the data obtained from the Western blots (compare Fig. 3.8.A and B) Unfertilised but aged eggs had a level of staining of a similar intensity to that of freshly-ovulated eggs (not shown). Mean pixel intensities taken from projected images of 5 unfertilised and fertilised oocytes confirmed the difference in staining intensity (62 ± 1 2 and 37 ± 6, respectively; t- test: P < 0.01) (Fig. 3.8.D).

3.7.3 Type I InsPgR expression is dow nregulated over the four hours follow in g

sperm fu sio n

I had so far demonstrated that type I InsP^Rs decrease in level within 8 hours of exposure to sperm. Since fusion of the sperm with the egg takes place very quickly in the absence of a zona pellucida this suggests that downregulation occurs within 8 hours of fertilisation itself. To determine whether the decrease correlates with any of the events of egg activation the time-course of the decrease in immunoreactivity was examined with greater accuracy. Oocytes were incubated for 15 minutes with capacitated sperm and returned to culture for 1-8 hours. At various time points throughout the culture period fertilised oocytes were removed and frozen in preparation for Western analysis.

The subsequent Western blots revealed that by 2 hours after exposure to sperm the levels of InsP)R had decreased to approximately half the control levels. By 4 hours the levels were 20-30% and by 6 hours the maximal decrease to 20% of control levels of immunoreactivity had been achieved (Fig. 3.9.A and B ). A separate blot confirmed that there was no significant further decrease in protein after 4 hours up until at least 7.5 hours post fertilisation (not shown). This indicates that the loss of InsP^R protein occurs gradually over the 4 hours following fertilisation and that the protein level then plateaus at this lower level.