Delineation of downstream signalling components during acrosome reaction mediated by heat solubilized human zona pelluci
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RESEARCH
Open Access
Delineation of downstream signalling components during acrosome reaction mediated by heat solubilized human zona pellucida Beena Bhandari1, Pankaj Bansal1, Pankaj Talwar2, Satish K Gupta1*
Abstract Background: Human egg is enveloped by a glycoproteinaceous matrix, zona pellucida (ZP), responsible for binding of the human spermatozoa to the egg and induction of acrosomal exocytosis in the spermatozoon bound to ZP. In the present manuscript, attempts have been made to delineate the downstream signalling components employed by human ZP to induce acrosome reaction. Methods: Heat-solubilized human ZP (SIZP) was used to study the induction of acrosome reaction in capacitated human spermatozoa using tetramethylrhodamine isothiocyanate conjugated Pisum sativum agglutinin (TRITC-PSA) in absence or presence of various pharmacological inhibitors. In addition, intracellular calcium ([Ca2+]i) levels in sperm using Fluo-3 acetoxymethyl ester as fluorescent probe were also estimated in response to SIZP. Results: SIZP induces acrosomal exocytosis in capacitated human sperm in a dose dependent manner accompanied by an increase in [Ca2+]i. Human SIZP mediated induction of acrosome reaction depends on extracellular Ca2+ and involves activation of Gi protein-coupled receptor, tyrosine kinase, protein kinases A & C and phosphoinositide 3 (PI3)- kinase. In addition, T-type voltage operated calcium channels and GABA-A receptor associated chloride (Cl-) channels play an important role in SIZP mediated induction of acrosome reaction. Conclusions: Results described in the present study provide a comprehensive account of the various downstream signalling components associated with human ZP mediated acrosome reaction.
Background Zona pellucida (ZP), a glycoproteinaceous matrix that surrounds the mammalian oocyte, plays an important role in species-specific binding of the spermatozoon to the oocyte, induction of acrosomal exocytosis in the ZPbound spermatozoa, avoidance of polyspermy and protection of the pre-implanted blastocyst. Human ZP matrix is composed of four glycoproteins designated as ZP1, ZP2, ZP3 and ZP4 whereas mouse ZP lacks ZP4 by virtue of it being a pseudogene. To accomplish fertilization, ZP mediated induction of acrosomal exocytosis is crucial that enables spermatozoa to penetrate the ZP matrix. In mouse, ZP3 is primarily responsible for induction of acrosome reaction [1,2] whereas in humans, ZP4 in addition to ZP3 contributes in * Correspondence: [email protected] 1 Reproductive Cell Biology Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi-110 067, India
induction of acrosome reaction [3-6]. Recent studies from our group suggest that in humans, ZP1 may also be involved in induction of acrosomal exocytosis (unpublished observations). It has also been proposed that a mechanosensory signal produced during zona penetration may also be required to initiate acrosome reaction [7]. At least, two different receptor mediated signalling pathways in sperm plasma membrane have been s
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