treated water; Sigma, Mississauga, ON, Canada) at 37uC for 5 min. The prehybridization performed by incubating the sections at 45uC for 60 min in the buffer containing 46 SSC, 10% dextran sulfate, 16 Denhardt’s solution (0.02% of FicollH 400, polyvinyl pyrolidone and bovine serum albumin), 2 mM EDTA, 50% deionized formamide, salmon sperm DNA (500 mg/ml); all purchased from Sigma, Mississauga. ON, Canada. Hybridization solution containing 100 ng/ml of digoxigenin-labeled PLCf riboprobes in the prehybridization solution was applied to each section and incubated in a moist chamber at 45uC overnight. Following hybridization, sections were washed twice with 26 SSC at 37uC for 5 min; four times with 60% formamide in 0.2% SSC at 37uC for 5 min and twice with 26 SSC at 37uC for 5 min. The sections were then blocked with 1% Blocking Reagent (Roche Diagnostics, Laval, QC, Canada) in 100 mM Tris-HCl pH 7.5, 150 mM NaCl at room temperature for 30 min, followed by incubation at room temperature for 3 h with 1:500 of an alkaline phosphatase-conjugated anti-digoxigenin antibody (Anti-Digoxi- genin-AP, Fab fragments, Roche Diagnostics, Laval, QC, Canada) diluted in the blocking solution. The slides were immersed in 100 mM Tris-HCl pH 7.5, 150 mM NaCl for 5 min and 100 mM Tris-HCl pH 9.5, 150 mM NaCl, 50 mM MgCl2, 10% polyvinyl alcohol (PVA) for 10 min. Sections were then immunostained at room temperature for 2 h in solution containing 34 mg/ml 4-nitroblue tetrazolium chloride (NBT), 18 mg/ml 5- bromo-4-chloro-3-indolyl-phosphate (BCIP) and 240 mg/ml le- vamisole (Sigma, Mississauga. ON, Canada) in 100 mM Tris-HCl pH 9.5, 150 mM NaCl, 50 mM MgCl2, 10% PVA. The sections were finally washed with TE buffer for 5 min, mounted in the mounting medium (VecaShield; Vector Labs, Burlingame, CA) and viewed by Zeiss AXIO Imager.A1 microscope with high numerical aperture objectives, and an AxioCam HRc camera operated by AxioVs40 V4.7 software (all Zeiss, Jena, Germany). Porcine Oocyte Collection and in Vitro Fertilization Pre-pubertal gilts from a local slaughterhouse were used for collection of ovaries and oocytes as described previously [70]. Oocytes were washed and transferred into TCM-199 medium containing 10 ng/ml epidermal growth factor, 0.5 mg/ml FSH, 0.5 mg/ml LH, 0.1% polyvinyl alcohol and 0.57 mM cysteine (mTCM-199) at 39uC in 5% CO2 atmosphere in humidified air. The oocyte-cumulus cell complexes were transferred to mTCM- 199 without FSH and LH after 22–24 h, followed by additional culture for 20 h under the same condition. Freshly ejaculated sperm-rich fraction was collected from a fertile boar as described previously [71]. Spermatozoa were re-suspended in modified Tris- References 1. Malter HE, Cohen J (2002) Intracytoplasmic sperm injection: technical aspects. In: Vayena E, Rowe PJ, Griffin PD, eds. Current Practices and Controversies in Assisted Reproduction. Geneva, Switzerland: World Health Organization. pp 126–133. 2. Dey SK (2010) How we are born. J Clin Invest 120: 952–955. 3. Centers for Disease Control and Prevention 2009 Assisted Reproductive Technology Report. Available: http://www.cdc.gov/art/artreports.htm Updat- ed December 2, 2011. Accessed 2012 Feb 5. 4. Andersen AN, Goossens V, Ferraretti AP, Bhattacharya S, Felberbaum R, et al. (2008) Assisted reproductive technology in Europe, 2004: results generated from European registers by ESHRE. Hum Reprod 23: 756–771. 5. Publicover S, Harper CV, Barratt C (2007) [Ca2+]i signalling in sperm–making the most of what you’ve got. Nat Cell Biol 9: 235–242. 6. Machaca K (2007) Ca2+ signaling differentiation during oocyte maturation. J Cell Physiol 213: 331–340. 7. Runft LL, Jaffe LA, Mehlmann LM (2002) Egg activation at fertilization: where it all begins. Dev Biol 245: 237–254. 8. Balakier H, Dziak E, Sojecki A, Librach C, Michalak M, et al. (2002) Calcium- binding proteins and calcium-release channels in human maturing oocytes, pronuclear zygotes and early preimplantation embryos. Hum Reprod 17: 2938–2947. Developmental Expression and Functions of PLCf buffered medium. IVF was performed with the final sperm concentration of 16106 cells/ml [70]. Fertilized oocytes were then cultured at 39uC in North Carolina State University (NCSU)-23 medium supplemented with 0.4% BSA in 5% CO2 atmosphere in humidified air. 6–8 h post-insemination, the oocytes were stripped of zona pellucida, fixed in 2% formaldehyde and permeabilized in 0.1% Triton X-100 to analyze the dynamics of PLCf at different stages of fertilization [72]. Oocytes were sequentially incubated with the primary and secondary antibodies for immunofluores- cence according to above mentioned protocol. The DNA stain DAPI (1:80) was mixed with secondary antibodies [65]. Supporting Information Figure S1 Control sections for Fig. 2 where primary antibody was preincubated with oligopeptides used to raise the immune serum before incubation with tissue section. Little to no immunostaining is present except a weak background staining in the interstitium, documenting the specificity of the primary antibody for the mouse (A) and human (B) spermatids. r, round spermatids. Bars = 20 mm. (TIF) Figure S2 Localization of PLCf in bull mature sperma- tozoa by immunofluorescence. Bull spermatozoa were fixed and processed for immunofluorescence as described previously [66]. The PLCf immunofluorescence was detected in post acrosomal region of permeabilized spermatozoa but not in NP40 extracted spermatozoa, similar to our findings in human and mouse. To confirm our results on NP40 extracted spermatozoa, we used anti-bull PAWP antibody as a control. PAWP is localized to sperm PAS and consequently is resistant to NP40. The results shown here are with anti-EF antibody. DNA staining was performed with DAPI, DIC; differential interference contrast. Bars = 5 mm. (TIF) Acknowledgments We would like to thank Ms. Judith Vanhorn and Ms. Miriam Sutovsky for their kind technical assistance. Author Contributions Conceived and designed the experiments: MA PS RO. Performed the experiments: MA YY WX YJY MT. Analyzed the data: MA SP PS RO. Contributed reagents/materials/analysis tools: MT SP PS RO. Wrote the paper: MA RO. 9. Sutovsky P, Manandhar G, Wu A, Oko R (2003) Interactions of sperm perinuclear theca with the oocyte: implications for oocyte activation, anti- polyspermy defense, and assisted reproduction. Microsc Res Tech 61: 362– 378. 10. 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J Biol Chem 282: 12164– 12175. PLoS ONE | www.plosone.org 10 March 2012 | Volume 7 | Issue 3 | e33496