Molecular Human Reproduction Vol.11, No.7 pp. 523–529, 2005 Advance Access publication July 22, 2005

doi:10.1093/molehr/gah188

Protein tyrosine phosphorylation, hyperactivation and progesterone-induced acrosome reaction are enhanced in IVF media: an effect that is not associated with an increase in protein kinase A activation F.L.C.Moseley1,2, K.N.Jha3, Lars Björndahl2, I.A.Brewis4, S.J.Publicover5, C.L.R.Barratt1,2 and L.Lefièvre1,2,6 1

Reproductive Biology and Genetics Group, Division of Medical Sciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK, 2Assisted Conception Unit, Birmingham Women’s Hospital, Birmingham B15 2TG, UK, 3Centre for Research in Contraceptive and Reproductive Health, Department of Cell Biology, University of Virginia, Charlottesville, VA 22908, USA, 4Department of Medical Biochemistry and Immunology, Cardiff University, Cardiff, CF14 4XN, UK and 5School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.

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To whom correspondence should be addressed at: Reproductive Biology and Genetics Group, University of Birmingham, Institute of Biomedical Research, Edgbaston, Birmingham, B15 2TT, UK. E-mail: [email protected] Sperm capacitation is a prerequisite for successful in vitro fertilization (IVF) and therefore a focus of sperm preparation in IVF laboratories. The technology of IVF is, therefore, potentially valuable in advancing our understanding of the molecular processes that occur during sperm capacitation. We have investigated sperm capacitation induced by a commercial IVF medium compared to that occurring in standard capacitating medium (CM) typically used in a nonclinical setting. Percoll-washed spermatozoa were resuspended in Cook® Sydney IVF medium, Cook® Sydney IVF sperm buffer, Earle’s balanced salt medium (capacitating medium) or a modified Earle’s balanced salt medium [non-capacitating medium (NCM)] for up to 120 min at 37ºC and, if applicable, in the presence of 5% CO2 in air. Sperm protein kinase A (PKA) activity, PKA-dependent serine/threonine phosphorylation, tyrosine phosphorylation, hyperactivation and progesterone-induced acrosome reaction were evaluated. IVF medium was shown to accelerate sperm capacitation (compared with capacitating medium) as determined by tyrosine phosphorylation, sperm hyperactivation and progesterone-induced acrosome reaction. This effect was not associated with enhanced activation of PKA or increased levels of serine/threonine phosphorylation. In contrast, IVF sperm buffer (used for sperm preparation) did not stimulate sperm capacitation when incubated for up to 90 min. We have shown that different capacitating media vary strikingly in their efficacy and that this difference reflects activation of a pathway other than the well-characterized activation of soluble adenylyl cyclase/cAMP/PKA. Key words: acrosome reaction/hyperactivaton/PKA-dependent serine/protein kinase A/threonine phosphorylation/tyrosine phosphorylation

Introduction After ejaculation, mammalian sperm undergo many biochemical and functional changes that render the cells competent to fertilize an oocyte, a process termed capacitation (de Lamirande et al., 1997; Visconti et al., 2002). The modifications observed in human spermatozoa include an efflux of cholesterol from the plasma membrane (Cross, 1998; Osheroff et al., 1999), increased activity of adenylyl cyclase, elevation of cyclic adenosine monophosphate (cAMP) and stimulation of protein kinase A (PKA) (Parinaud and Milhet, 1996; Chen et al., 2000; Lefièvre et al., 2002), increased protein serine/threonine and tyrosine phosphorylation (Leclerc et al., 1996; Osheroff et al., 1999; O’Flaherty et al., 2004), changes in [Ca2+]i (Baldi et al., 1991) and events involving components of the extracellular signal-regulated kinase (ERK) family of mitogen-activated protein kinases (MAPK) (Luconi et al., 1998; de Lamirande and Gagnon, 2002). Functional changes associated with this include alterations in the pattern of motility (termed hyperactivation) and acquisition of the ability to undergo the acrosome reaction

in response to physiological or non-physiological agonists (de Lamirande et al., 1997). Human sperm capacitation can be induced during in vitro incubation. Many well-characterized incubation media have been shown to support capacitation [Tyrodes, Hams-F10, Biggers, Whitten and Whittingham (BWW), supplemented Earle’s balanced salt solution (sEBSS), Hank’s balanced salt solution (HBSS) and Synthetic Human Tubal Fluid (HTF)] (Edwards et al., 1980; Mortimer, 1986; Calvo et al., 1993) as well as supplementation of media with uncharacterized biological fluids [fetal cord serum ultrafiltrate (FCSu), follicular fluid (FF), follicular fluid ultrafiltrate (FFu)] and progesterone (found in the female tract). It is not known whether all these treatments induce capacitation through the same signalling pathways or with the same degree of efficacy. In addition to the wide range of capacitating media used, the conditions used by different laboratories for in vitro capacitation vary in both the sperm concentration used and, in particular, the period for which cells are incubated (ranging from 2 to 24 h). These are important factors to

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F.L.C.Moseley et al. take into account. For example, it has been reported that short co-incubation of gametes (1–2 h) during in vitro fertilization (IVF) significantly improves embryo quality in comparison with longer incubations (20 h) (Quinn et al., 1998; Dirnfeld et al., 1999; Kattera and Chen, 2003). Prolonged co-incubation of gametes appears to produce high levels of reactive oxygen species that are detrimental to sperm motility and viability (de Lamirande and Gagnon, 1995) and induce zona hardening for the oocyte as well as affecting embryo viability (Gianaroli et al., 1996). The discovery of sperm capacitation, independently by Chang (1951) and Austin (1952), was essential for the development of techniques and media for in vitro preparation of fertilization-competent human spermatozoa (Bavister, 2002). During the development of IVF methodologies, specialized fertilization media have been developed on the basis of their efficacy and reliability in the IVF clinic. IVF medium, which is known to support fertilization leading to pregnancy and live births, is now commercially available, standardized and widely used. Experimental studies on the dynamics of capacitation in an IVF system (induction of sperm capacitation using an IVF media) are thus potentially of great value in identifying the important functional changes required for successful fertilization. Surprisingly, the methods of sperm preparation for IVF have not been exploited to gain a better understanding of the processes leading to sperm capacitation. Capacitation-related markers have not been closely studied in cells prepared using an IVF system and only recently it has been shown that low levels of tyrosine phosphorylation observed on the sperm flagellum (