The Testicular and Epididymal Expression Profile of PLCf in Mouse and Human Does Not Support Its Role as a Sperm-Borne Oocyte Activating Factor Mahmoud Aarabi1,2, Yang Yu 1 , Wei Xu 1 , Man Y. Tse1, Stephen C. Pang 1 , Young-Joo Yi3, Peter Sutovsky3,4, Richard Oko 1 * 1 Department of Biomedical and Molecular Sciences, School of Medicine, Queen’s University, Kingston, Ontario, Canada, 2 Department of Genetics, Reproductive Biotechnology Research Center, Avicenna Research Institute, Tehran, Iran, 3 Division of Animal Sciences, University of Missouri, Columbia, Missouri, United States of America, 4 Departments of Obstetrics, Gynaecology and Women’s Health, University of Missouri, Columbia, Missouri, United States of America Abstract Phospholipase C zeta (PLCf) 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 PLCf, 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 PLCf by immunoblotting. In both human and mouse, the active isoform of PLCf was detected in sperm fractions other than PT, where SOAF is expected to reside. Developmentally, PLCf 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 PLCf 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, PLCf 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 PLCf 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 PLCf 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 PLCf is incorporated into the detergent-resistant perinuclear theca fraction where SOAF resides. Citation: Aarabi M, Yu Y, Xu W, Tse MY, Pang SC, et al. (2012) The Testicular and Epididymal Expression Profile of PLCf in Mouse and Human Does Not Support Its Role as a Sperm-Borne Oocyte Activating Factor. PLoS ONE 7(3): e33496. doi:10.1371/journal.pone.0033496 Editor: Hugh Clarke, McGill University, Canada Received January 9, 2012; Accepted February 13, 2012; Published March 12, 2012 Copyright: 2012 Aarabi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This project was supported by Canadian Institutes of Health Research Grant No. MOP-84440 to R.O., National Research Initiative Competitive Grants No. 2007-35203-18274 and No. 2011-67015-20025 from the United States Department of Agriculture (USDA) National Institute of Food and Agriculture, to P.S., and by the Food for the 21st Century Program of the University of Missouri-Columbia to P.S. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: ro3@queensu.ca Introduction Assisted Reproductive Technologies (ART) including in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) provide treatment options for infertile couples; a problem that is estimated to affect 15% of world population [1,2]. While over 146,000 ART cycles were performed in United States in 2009, most of these cycles (63%) did not produce pregnancy [3], similar to results from European countries [4]. Failure of ART to conceive babies could be partially due to sperm inability to induce intracellular calcium release and resumption of second meiosis in the oocyte, a process called oocyte activation [5]. Oocyte activation includes the sperm-induced molecular and cellular events that occur during the transition from haploid gametes to a diploid zygote [6]. It is well established that oocyte activation is initiated after sperm delivers a testis-specific, sperm-borne oocyte activating factor (SOAF) into the ooplasm [7]. However, it is still unclear what sperm protein(s) play this crucial role. To be considered as SOAF, a candidate protein should meet specific developmental and functional criteria. Microinjection of SOAF complementary RNA (cRNA) or recombinant protein into the oocyte is expected to mimic the oocyte activation induced by ICSI. This effect includes the induction of calcium oscillations in mammalian metaphase II arrested oocytes followed by formation of secondary polar body and a female pronucleus [6]. Moreover, relevant antibodies and competitive peptides derived from a SOAF candidate should block the sperm induced oocyte activation during ICSI. SOAF should reside and remain, during early stages of fertilization, in the postacrosomal sheath [8] of sperm perinuclear theca (PAS-PT), a detergent- resistant layer surround- ing sperm nuclei [9]. In order to be a PAS-PT component a SOAF candidate should originate during spermatid elongation [10]. Because of potential benefits of SOAF in the diagnosis and treatment of male infertility, it has been highly sought after. Several candidates have been considered in the past [11,12,13], PLoS ONE | www.plosone.org 1 March 2012 | Volume 7 | Issue 3 | e33496