Phospholipase C zeta undergoes dynamic changes in its pattern of localization in sperm during capacitation and the acrosome reaction  Claire Young, B.Sc., Patricia Grasa, Ph.D., Kevin Coward, Ph.D., Lianne C. Davis, B.Sc., John Parrington, Ph.D.  Fertility and Sterility  Volume 91, Issue 5, Pages 2230-2242 (May 2009) DOI: 10.1016/j.fertnstert.2008.05.021 Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Multiple sequence alignment of hamster, mouse, and chicken PLCζ proteins. Identical residues indicated by black shading, conserved residues by dark grey shading, and similar residues by light grey shading. †Catalytically important residues; ‡residues vital for Ca2+ binding; #residues forming a putative phosphoinoside binding site. Small bold dots represent EF Hand, X and Y domains, and C2 domain. Large black dots indicate positions of peptides used to create the polyclonal antibody. Fertility and Sterility 2009 91, 2230-2242DOI: (10.1016/j.fertnstert.2008.05.021) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Dendrogram of PLCζ, and PLCδ1 sequences. The topology of this dendrogram is supported by boostrap values of 100% at all internal nodes. Fertility and Sterility 2009 91, 2230-2242DOI: (10.1016/j.fertnstert.2008.05.021) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 3 (A) Tissue expression of hamster PLCζ as detected by RT-PCR. Target PCR product is 500 bp. (B) Northern hybridization demonstrating tissue specificity of hamster PLCζ. (C) Northern hybridization demonstrating developmental expression of hamster PLCζ at 5, 10, 17, 30, 63 days after birth. T = testis; L = liver; H = heart; K = kidney; Lu = lung; -ve = negative control. Fertility and Sterility 2009 91, 2230-2242DOI: (10.1016/j.fertnstert.2008.05.021) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 4 (A) Immunoblot with anti-PLCζ antibody demonstrating detection of recombinant mouse GST-PLCζ (approximately 100 kDa). (B) Immunoblot blot with anti-PLCζ antibody demonstrating presence of immunoreactive bands indicative of PLCζ at approximately 74 kDa in (1) mouse sperm and (2) hamster sperm. (C) Immunoblot of untreated intact mice sperm (lane 1) and the soluble fraction obtained after incubating mouse sperm with Triton X-100 (which should correspond to the acrosomal PLCζ population) (lane 2), and Triton extracted sperm (which should contain the post-acrosomal PLCζ population) (lane 3). An expected protein band of approximately 74 kDa was observed in intact sperm and was also present in the Triton-extracted sperm (lanes 1 and 3), in line with the post-acrosomal PLCζ population corresponding to full length PLCζ. However, full-length PLCζ was not detected in the Triton-soluble fraction (lane 2). Instead we observed a new prominent protein band at approximately 55–60 kDa (asterisk) in this fraction that is not obvious in either of the other two samples. Fertility and Sterility 2009 91, 2230-2242DOI: (10.1016/j.fertnstert.2008.05.021) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 5 Immunofluorescent studies in (A–D) non-capacitated, (E–H) capacitated, and (I–L) ionophore-treated mouse sperm. (M–P) Secondary antibody alone. (A, E, I, M) Anti-PLCζ immuofluorescence. (B, F, J, N) FITC-PNA-lectin staining identifying acrosome. (C, G, K, O) Sperm nucleus stained with Hoescht 33342. (D, H, L, P) Merged fluorescent and brightfield data. (Q, R) Merged fluorescent and brightfield PLCζ immunolocalization, in (Q) absence and (R) presence of immunogenic peptide. Arrows indicate position of positive PLCζ immunoreactivity. Original photographs taken at ×63 magnification with ×3.5 digital zoom. Fertility and Sterility 2009 91, 2230-2242DOI: (10.1016/j.fertnstert.2008.05.021) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 6 Immunofluorescent studies in (A–D) non-capacitated, (E-H) capacitated, and (I–L) ionophore-treated hamster sperm. (M–P) Secondary antibody alone. (A, E, I, M) Anti-PLCζ immuofluorescence. (B, F, J, N) FITC-PNA-lectin staining identifying acrosome. (C, G, K, O) Sperm nucleus stained with Hoescht 33342. (D, H, L, P) Merged fluorescent and brightfield data. (Q, R) Merged fluorescent and brightfield PLCζ immunolocalization, in (Q) absence and (R) presence of immunogenic peptide. Arrows indicate position of positive PLCζ immunoreactivity. Original photographs taken at ×63 magnification with ×3.5 digital zoom. Fertility and Sterility 2009 91, 2230-2242DOI: (10.1016/j.fertnstert.2008.05.021) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 7 Immunofluorescent studies in non-capacitated (A–D) mouse and (M–P) hamster, capacitated (E–H) mouse and (Q–T) hamster, and ionophore-treated (I–L) mouse and (U–X) hamster sperm treated with Triton X-100 before fixing. (A, E, I, M, Q, U) Immunofluorescent images of PLCζ localization. (B, F, J, N, R, V) FITC-PNA-lectin staining indicating absence of acrosome in these samples. (C, G, K, O, S, W) Hoescht 33342 staining of sperm nucleus. (D, H, L, P, T, X) Merged fluorescent and brightfield data. Arrows indicate position of positive PLCζ immunoreactivity. Original photographs taken at ×63 magnification with ×3.5 digital zoom. Fertility and Sterility 2009 91, 2230-2242DOI: (10.1016/j.fertnstert.2008.05.021) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions