PLCy1, PLCy2, and PLCS4 and/or Its Splice Variants Are Not the Active Components of SF
Because injection of SF triggers IP3 production, it may be that the sperm’s Ca2+ releasing component is a PLC. To investigate if some of the known isoforms of PLC may represent the Ca2+ activating signal of SF, we first fractionated pig testis extracts and pig SF by Superose 12, hydroxy-apatite FPLC, and/or ammonium sulfate precipitation and then tested the Ca2+ releasing activity and the presence of PLCs in these fractions. Injection of unfractionated pig testis extracts (n = 10) into mouse eggs induced [Ca2+]i oscillations that closely resembled those initiated by fertilization or triggered by SF injection (Fig. 4A). Also, among the testis extracts Superose 12 and hydroxyapatite fractions, fraction 4 (Fig. 4B; Superose 12) and fractions 5 and 6 (Fig. 5, A and B; hydroxyapatite) exhibited Ca2+ releasing activity, which were the same fractions that showed Ca2+ releasing activity following Superose 12 and hydroxyapatite fractionation of SF preparations.
These data suggest that the Ca2+ releasing protein(s) in testis extracts has a size and phosphate binding ability similar to that of SF and, most likely, is the same protein(s). Western blots of Superose 12 fraction 4 from testis extracts showed that this fraction did not contain either PLC71 or PLC72 (Fig. 4C). In contrast, testis fractions that contained enhanced amounts of PLC71, PLC72, or both PLC7 isoforms such as fraction 3 showed no Ca2+ releasing activity (Fig. 4, B and C).
FIG. 4. Chromatographic fractionation of pig testis (PT) extracts and SF. Injection of pig testis extracts (5 mg/ml) into mouse eggs induced [Ca2+] oscillations (A). Pig testis extracts were fractionated using Superose 12 chromatography, and the fractions were pooled, concentrated, and tested for Ca2 + oscillation-inducing activity (B) and presence of PLC^1 and PLC^2 (C). Co, Cell lysate from A431 cells that was used as positive control (provided by Upstate Biotechnology). SF was fractionated by hydroxyapatite (HA) chromatography (D), and each fraction was tested for the presence of PLC^1 and Ca2+ oscillation-inducing activity. CE, Bovine cumulus cell extracts. The active fraction F3 from the HA column (SF-HA) was further fractionated by Superose 12 (E). Each fraction was tested for the presence of PLC^2 and Ca2+ oscillation-inducing activity. The expected position of full-size PLC^s is denoted by an arrowhead; 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.