Developmental Expression and Functions of PLCf Figure 5. PLCf mRNA and protein expression in mouse testis and epididymis. (A) RT-PCR detection of PLCf mRNA (mPLCf) in mature testis (M Tes.) and epididymis (M Ep.) of an adult mouse. mPLCf was also detected in the 4 week old mouse epididymis (4w Ep.) excluding the possible contamination with RNA from testicular sources (4w Tes.). Primers were specifically designed to detect the full size open reading frame of mPLCf. Utilizing second nested PCR, the results were confirmed by primers designed to detect the sequence encoding PLCf EF hand domain (mPLCf-EF). Mouse GAPDH was used as the house keeping gene for optimization of PCR results. (B) In situ hybridization of PLCf mRNA on paraffin embedded mouse epididymal tissue. Inset shows perinuclear localization of mRNA within the principal cell. The interstitium within the inset is marked by asterisk. No signal is detected in the corresponding regions of control probe (right panel). (C) Paraffin embedded mouse epididymal tissues immunoproxidase stained with anti-EF antibody. Similar results were obtained by anti-hmPLCf antibody. High level of PLCf expression was detected in the apical region (arrowheads) of principal cells, where secretory vesicles accumulate and the supranuclear region of Golgi apparatus. Right panel shows the result of pre-incubating the primary antibody with the oligopeptides used to raise the immune serum. Bars = 25 mm Bar in the inset: 5 mm. doi:10.1371/journal.pone.0033496.g005 embedded mouse epididymal tissue. PLCf immunostaining was detected in the principal cells of the epididymis, with the reaction product being most concentrated in the apical and supranuclear regions indicating Golgi apparatus contribution (Fig. 5C). The principal cells within initial segment of the caput epididymis were found to be most immunoreactive. Within the epididymal lumen, immunostaining was most evident in sperm tail and cytoplasmic droplet (not shown). PLCf depletion after acrosome reaction during porcine in vitro fertilization To answer whether the localization of PLCf on the sperm head is appropriate for its suggested role as SOAF, the fate of boar sperm PLCf during in vitro fertilization was followed. At the time of sperm-zona pellucida contact, PLCf immunofluorescence pre- dominated in the acrosomal region (Fig. 6). Subsequently, during the zona-induced acrosome reaction, the immunoreactivity accompanied the acrosomic shroud and was lost from the sperm head. After zona penetration and during sperm-oolemma binding and sperm incorporation into the oocyte cytoplasm, the PLCf signal was no longer detectable (Fig. 6). Discussion During fertilization, motile and fertilization competent sperma- tozoa pass through the female reproductive tract, interact with the zona pellucida and activate the metaphase II arrested oocytes after sperm-oolemma fusion [34]. The ability of spermatozoa to activate oocytes depends upon the normal expression of the sperm borne PLoS ONE | www.plosone.org 5 March 2012 | Volume 7 | Issue 3 | e33496