. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. 184 M. SAIGLISA AND H. IWASAKI Days before hatching 14d 10d 6d 4d 2d 1d 4h Z REM — 32 kDa -> kDa Figure 8. The appearance of immunoreactive OHSS in developing crab embryos. Embryo clusters (one- third of an ovigerous seta) were detached from a single female, crushed, and then denatured with the lysis buffer. The extracts (supernatant) were subjected to SDS-PAGE, and blots were stained immunochemically with the polyclonal OHSS antiserum. d (or /?): days (or hours) before hatching. Z: post-hatched embryo
. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. 184 M. SAIGLISA AND H. IWASAKI Days before hatching 14d 10d 6d 4d 2d 1d 4h Z REM — 32 kDa -> kDa Figure 8. The appearance of immunoreactive OHSS in developing crab embryos. Embryo clusters (one- third of an ovigerous seta) were detached from a single female, crushed, and then denatured with the lysis buffer. The extracts (supernatant) were subjected to SDS-PAGE, and blots were stained immunochemically with the polyclonal OHSS antiserum. d (or /?): days (or hours) before hatching. Z: post-hatched embryos (zoeas) released from the same female. REM: remnant structures remaining attached to the female's ovigerous hairs after hatching. Numbers to the left are the molecular masses of the same markers shown in Figure 2B Note that only the 32-kDa band is delected in the remnant structures (see Fig. 7). 32-kDa protein band was detected in these remnants (Fig. 8). Furthermore, a strong OHSS activity was also detected in these remnants (Table I), as well as in the supernatant after treatment with detergent (Table II). Hence, we specu- late that the 32-kDa band is an active OHSS. On the other hand, we cannot yet hypothesize that the 30-kDa protein band also has OHSS activity. Since purified antibodies recognized both the 32-kDa and 30-kDa bands (Figs. 6A and 6B). we suppose that these two proteins have very similar sequences; , the bands detected by immu- nochemical staining are probably both OHSS-related pro- teins. The OHSS antiserum detected not only 32-kDa and 30- kDa proteins, but also a band at 55 kDa (Figs. 8 and 9). When the active fractions 6 and 7 of molecular-sieve chro- matography were studied (Fig. 3A). this band was not detected at all. In contrast, when the crushed embryos (Figs. 8 and 9) were examined, the 55-kDa band often appeared clearly; but it did not appear in the remnant (Fig. 7). The 55-kDa band also appeared on molecular-sieve chromatog- raphy (Fig. 2B), b
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