Developmental Expression and Functions of PLCf Figure 2. Developmental localization of PLCf in mouse (A) and human (B, C) testes utilizing anti-hmPLCf antibody. Similar results were obtained with anti-EF antibody in mouse and human and anti-hPLCf in human sections. (A) Immunostaining originates at the beginning of acrosome formation. In stage III it appears confined to the acrosomic vesicle (AV) of step 3 spermatids as seen in detail in the inset; note that acrosomic granule is most immunoreactive. In stage VII immunostaining is intense within the acrosome, capping a portion of the nucleus (arrowheads) of the step 7 round spermatids as seen in more detail in the inset. In stages XI and XII immunostaining resides in the head of elongating spermatids in steps 11 and 12, respectively (arrows). However, by step 14 (stages II and III) the intensity of immunostaining in elongated spermatid heads has diminished signif- icantly (arrows). (B, C) In human, there are six stages (I–VI) of the cycle of the seminiferous epithelium. As seen in B, PLCf accumulates over the acrosomic vesicle (AV) of step 2 spermatids in stage II. Little immunoreactivity is found elsewhere in the epithelium and the elongated spermatids (arrowheads) appear unreactive. In step 4 spermatids (stage IV, see C), the fully formed acrosome is intensely labeled (arrows). Bars = 20 mm; Bars in insets = 5 mm. doi:10.1371/journal.pone.0033496.g002 Immunofluorescence localization of sperm PLCf Immunogold and immunoperoxidase developmental data demonstrating PLCf’s acrosomal origin were reinforced by immunofluorescence performed on testicular germ cell spreads. As shown in Fig. 4A, PLCf immunofluorescence was exclusive to the acrosome of mouse round and elongating spermatids. The predominance of PLCf in the acrosome of spermatids suggested that the detergent soluble PLCf-reactive band(s) detected in westerns of spermatozoa (Fig. 1) were most likely of acrosomal origin. However, the predominant immunofluorescence labelling of PLCf in mouse cauda-epididymal spermatozoa was over the postacrosomal region of the sperm head (Fig. 4B). In humans, PLCf also appeared to be localized over both postacrosomal and acrosomal regions (Fig. 4C). Because of this unexpected localiza- tion pattern after epididymal passage, the possibility of epididymal secretion and sperm surface acquisition of PLCf was investigated. Figure 3. LR-White embedded mouse testicular sections immunogold labeled with affinity purified anti-hmPLCf anti- body. Similar results were obtained by anti-EF. (A) Section through portion of Step 1 spermatid showing Golgi complex (GC), containing several proacrosomic granules (PG) that are immunoreactive for PLCf. (B) By step 2, several immunoreactive proacrosomic granules have fused with each other forming the acrosomic vesicle (AV), which attaches to the spermatid nucleus (N). (C) Section through a portion of step 3 spermatid showing the immunogold labeled AV docked onto the nucleus with the subacrosomal layer (SL) intervening. Underlying the nuclear envelope in the region of the forming acrosome is the electron dense nuclear lamina (outlined by asterisks). (D) As acrosome capping proceeds over the apical half of the nucleus in step 6 spermatids a high concentration of immunogold labeling is evident in the expanding acrosomal collar (AC) coincident with a loss of labelling in the acrosomic granule. (E) Step 16 spermatids, almost fully formed, show a diminution of labeling in the acrosome and no labeling is detected in the perinuclear theca. AS, apical segment of acrosome; ES, equatorial segment of acrosome; GC, Golgi complex; GS, Golgi saccules; N, nucleus; PAS, postacrosomal sheath; SM, Sertoli cell mantle. Bars = 0.2 mm. doi:10.1371/journal.pone.0033496.g003 Comparing permeabilized vs. non-permeabilized spermatozoa, the immunofluorescence was similar suggesting localization of PLCf to the sperm surface. Furthermore, removal of the plasma membrane-bound proteins and exposure of PAS-PT by NP40 eliminated PLCf fluorescence. Complementary to our findings in mouse and human, PLCf immunofluorescence in bull spermato- zoa was localized to the postacrosomal region and was removable by NP40 extraction prior to fixation (Fig. S2). Expression of PLCf in mouse epididymis The finding that PLCf localized to the cauda sperm head surface led to a hypothesis that PLCf is not a strictly testis specific molecule as dogma would suggest, but also an epididymal secretory protein. To test this hypothesis, semi-quantitave reverse transcriptase polymerase chain reaction (RT-PCR), using specific PLCf primers, was performed on total mRNA isolated from mature mouse testes and epididymides, revealing PLCf mRNAs of similar size in both tissues. To avoid contamination of epididymal samples with testicular spermatozoa, tissues from 28 day old mice were also tested, providing a similar result to that found in adult PLoS ONE | www.plosone.org 3 March 2012 | Volume 7 | Issue 3 | e33496