Some of the smaller size bands recognized by the antibodies in the active fraction, upon further fractionation, were segregated into inactive fractions (data not shown). Similarly, fractionation of SF by hydroxyapatite column followed by Western blotting revealed that active fractions F3 and F4 did not contain immunoreactive PLC71 (Fig. 4D); bovine cumulus cell extracts, which contained full size PLC71, did not exhibit Ca2+ oscillation-inducing activity.
PLC72 in SF, whose presence was tested after sequential hydroxyapatite and Superose 12 chromatography, was not detected in fraction F4, which had maximal activity (Fig. 4E). In addition, studies using mouse SF, which contained full size PLC7I and PLC72, demonstrated that the Ca2+ releasing activity of these fractions was unaffected by immunodepletion of both PLC7 isoforms (data not shown).
Recently, it has been shown that two splicing variants of PLC84, ALTI and ALTII, are abundantly expressed in rat testis. To determine whether PLC84 and/or its splice variants represent the active component in our fractions, hydroxyapatite fractionation of pig testis extracts was carried out. As shown in Figure 5C, Western blotting of fractions 5-7, which were the active fractions, did not reveal the presence of PLC84 and/or its splice variants, although a strongly reacting band was detected in the unfractionated testis extract and in inactive fraction 2. In addition, ammonium sulfate precipitation of pig SF followed by Western blotting revealed that a band with an approximate molecular mass of 90 kDa, presumably one of the splice variants of PLC84, was segregated into the supernatant, which lacked Ca2+ releasing activity (Fig. 5D). Together, these results suggest that PLC7I, PLC72, and PLC84 and/or its splice variants are not likely to be responsible for the Ca2+ releasing activity of SF. The possibility that other PLC isoforms constitute the active component in SF was also investigated. However, because of the cross-reactivity of the commercially available antibodies, it was impossible to discern the contribution of any of these isoforms to the Ca2+ releasing activity of SF.
FIG. 5. Hydroxyapatite fractionation of pig testis (PT) extracts and ammonium sulfate fractionation of pig SF. Hydroxyapatite fractions (A) were pooled, concentrated, and tested for Ca2 + releasing activity (B) and presence of PLCS4 and/or its splice variants (C). Crude SF was fractionated by ammonium sulfate precipitation (D). Precipitates (P) and supernatants (SN) obtained by exposure to 50% saturating solutions were tested for the presence of PLCS4 and for Ca2 + oscillation-inducing activity. The arrowhead denotes the expected position of PLCS4 and/or its splice variants; 20 ^g of total protein was loaded per lane. At least two eggs per fraction were injected each time to test Ca2+ activity. Injections were repeated at least three different times. Arrow denotes time of injection.