The testicular and epididymal expression profile of PLCζ in mouse and human does not support its role as a sperm-borne oocyte activating factor

Abstract

Phospholipase C zeta (PLCζ) is a candidate sperm-borne oocyte activating factor (SOAF) which has recently received attention as a potential biomarker of human male infertility. However, important SOAF attributes of PLCζ, including its developmental expression in mammalian spermiogenesis, its compartmentalization in sperm head perinuclear theca (PT) and its release into the ooplasm during fertilization have not been established and are addressed in this investigation. Different detergent extractions of sperm and head/tail fractions were compared for the presence of PLCζ by immunoblotting. In both human and mouse, the active isoform of PLCζ was detected in sperm fractions other than PT, where SOAF is expected to reside. Developmentally, PLCζ was incorporated as part of the acrosome during the Golgi phase of human and mouse spermiogenesis while diminishing gradually in the acrosome of elongated spermatids. Immunofluorescence localized PLCζ over the surface of the postacrosomal region of mouse and bull and head region of human spermatozoa leading us to examine its secretion in the epididymis. While previously thought to have strictly a testicular expression, PLCζ was found to be expressed and secreted by the epididymal epithelial cells explaining its presence on the sperm head surface. In vitro fertilization (IVF) revealed that PLCζ is no longer detectable after the acrosome reaction occurs on the surface of the zona pellucida and thus is not incorporated into the oocyte cytoplasm for activation. In summary, we show for the first time that PLCζ is compartmentalized as part of the acrosome early in human and mouse spermiogenesis and is secreted during sperm maturation in the epididymis. Most importantly, no evidence was found that PLCζ is incorporated into the detergent-resistant perinuclear theca fraction where SOAF resides.

Figure 1. Immunoblotting of PLCζ after differential extraction of mouse and human spermatozoa.

(A) Detergent extraction of mouse cauda epididymal spermatozoa. The ∼74 kDa band corresponding to the functional isoform of PLCζ (arrow) was detected in whole sperm (WS) and was resistant to NP40 extraction. Anti-hmPLCζ was used to detect the signal with a similar result obtained with anti-EF. (B) The NP40 resistant sperm fraction (i.e., pellet in panel A) was sonicated and sperm heads and tails were separated through a sucrose gradient. The 74 kDa active isoform was found only in the tail fraction (arrow). Further incubation of the tail fraction with Triton x-100/DTT (Tail Tx-DTT) extracted all of the PLCζ bands, suggesting the mitochondrial sheath of the mid piece as the origin of PLCζ in the sperm tail. Anti-EF was used to detect the signal with a similar result obtained with anti- hmPLCζ. Lanes were run on the same gel but were non-contiguous. (C) Human sperm extraction by NP40. The functional isoform (arrow) was detergent extractable as shown in NP40 lane. Anti-hmPLCζ was used for detection. A similar result was observed with anti-EF and anti-hPLCζ. WS, Whole sperm; Sc.Sn, Sonication supernatant.

Figure 2. Developmental localization of PLCζ in mouse (A) and human (B, C) testes utilizing anti-hmPLCζ antibody.

Similar results were obtained with anti-EF antibody in mouse and human and anti-hPLCζ 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 significantly (arrows). (B, C) In human, there are six stages (I–VI) of the cycle of the seminiferous epithelium. As seen in B, PLCζ 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 µm; Bars in insets = 5 µm.

Figure 3. LR-White embedded mouse testicular sections immunogold labeled with affinity purified anti-hmPLCζ antibody.

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 PLCζ. (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 µm.

Figure 4. Localization of PLCζ in mouse testicular spermatids, and mouse and human mature spermatozoa by immunofluorescence.

(A) PLCζ (red) localized to the forming acrosome in round spermatid (top) and elongated spermatid (bottom) of testicular spreads. DNA was stained by DAPI (blue). (B, C) Mature spermatozoa were extracted from cauda epididymis and ejaculate in mouse and human, respectively. The PLCζ immunofluorescence was detected in both non-permeabilized and permeabilized mouse (B) and human (C) spermatozoa. NP40 extraction abolished the detected signal on the sperm head, confirming the presence of PLCζ on the surface of mature spermatozoa. Immunolocalization above was done with anti-EF with a similar result obtained with anti-hmPLCζ. DNA staining is with DAPI in A (blue) and SYTOX green in B (green), DIC; differential interference contrast. Bars = 5 µm.

Figure 5. PLCζ mRNA and protein expression in mouse testis and epididymis.

(A) RT-PCR detection of PLCζ mRNA (mPLCζ) in mature testis (M Tes.) and epididymis (M Ep.) of an adult mouse. mPLCζ 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 mPLCζ. Utilizing second nested PCR, the results were confirmed by primers designed to detect the sequence encoding PLCζ EF hand domain (mPLCζ-EF). Mouse GAPDH was used as the house keeping gene for optimization of PCR results. (B) In situ hybridization of PLCζ 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-hmPLCζ antibody. High level of PLCζ 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 µm Bar in the inset: 5 µm.

Figure 6. Indirect immunofluorescence analysis of PLCζ during in vitro fertilization in swine.

Note the PLCζ immunoreactivity (red) in the acrosome region during sperm-zona binding. The immunoreactivity disappears along with acrosome shroud (arrows) when the spermatozoa undergo the acrosome reaction. No PLCζ immunoreactivity was detectable on the sperm heads during sperm-oolemma binding and incorporation into the oocyte. The results shown here are from anti-pPLCζ while a similar result was observed with anti-EF. DAPI (blue) was used for DNA staining; DIC, Differential Interference Contrast. Bars = 25 µm.