Role of the major ecto‐phosphoprotein in sperm flagellar motility using a cell electroporation method

Abstract

Previous studies from our laboratory have identified MPS, a 100-kDa protein, as the major phosphoprotein substrate of caprine sperm ecto-cyclic AMP independent protein kinase. In this study the isolated (32)P-labelled MPS has been incorporated into mature caprine (Capra indicus) cauda-epididymal spermatozoa with the help of cell electroporation technique to investigate the effect of MPS on sperm flagellar motility. The optimum conditions for electroporation of sperm cells consisted of exposure of 0.2 ml of sperm cells (2 x 10(8)/ml) to external electric field of intensity 1.5 kV/cm and capacitation of 25 microF at 4 degrees C and post-pulse incubation at 37 degrees C for 1 hr. when nearly 50% of the cells lost motility. Scanning electron micrographs (SEM) demonstrate the formation of micro-pores and local osmotic swelling in the electroporated spermatozoa. MPS incorporation was maximal when its concentration was 30 microg/ml (300 pmol) in the medium and when the post-pulse incubation time was 60 min. At maximum (75%) MPS incorporation, total and forward motility increments were also maximum: 34% (P < 0.01) and 32% (P < 0.01), respectively. The subcellular fractionation data show that major portion of the introduced MPS was bound to the plasma-membrane of spermatozoa. The 32P-labelled electrophoresed intact spermatozoa lost radioactivity due to the action of the endogenous ecto-phosphoprotein phosphatase. Therefore MPS is primarily localised on the sperm external surface leaving its phosphate group(s) oriented in the extracellular medium. The data provided further evidence to strengthen the view that MPS is an ecto-phosphoprotein and that it plays an important role in the regulation of sperm flagellar motility.

Sperm ecto‐protein kinase and its protein substrate: Novel regulators of membrane fusion during acrosome reaction

“…Consequently the precise role of cytosolic protein kinases/phosphoproteins in the acrosome reaction is largely unknown. As elaborated in the “Introduction” Section, a novel protein kinase (CIK) and its endogenous membrane‐bound protein substrate (MPS) have been shown to occur on sperm head overlying the acrosome (Maiti et al, 2004, 2008; Nath et al, 2008). This study reports for the first time, the role of the well‐defined sperm surface ecto‐protein kinase and its phosphoprotein substrate in the regulation of acrosome reaction triggered by Ca ++ ionophore in vitro using the caprine sperm model.…”

“…Although earlier investigators have provided several lines of evidences for the occurrence of ecto‐protein kinases in a variety of mammalian cells (Mastro and Rosengurt, 1976; Schlaeger and Kohler, 1976; Majumder, 1981; Halder and Majumder, 1986; Dey and Majumder, 1990; Walter et al, 2000; Guthmann et al, 2002; for review Nath et al, 2008), the above‐mentioned findings from our laboratory provide confirmatory evidence for the localization of an ecto‐protein kinase and its protein substrate on a cell surface. The data demonstrate that ecto‐CIK through its substrate protein: MPS plays a vital role in the regulation of sperm forward progression and velocity (Maiti et al, 2004, 2008; Nath et al, 2008). MPS serves as a significant promoter of sperm forward progression.…”

“…The ecto‐protein kinase is primarily localized in the acrosomal cap area of the external surface of the mature sperm head as demonstrated by indirect immunofluorescence studies (Nath et al, 2008). “Ecto” nature of CIK as well as MPS has been established conclusively using indirect immunofluorescence studies based on antibodies of these pure antigens and studies on activities of these molecules utilizing their monovalent antibodies (Maiti et al, 2004, 2008; Nath et al, 2008). Although earlier investigators have provided several lines of evidences for the occurrence of ecto‐protein kinases in a variety of mammalian cells (Mastro and Rosengurt, 1976; Schlaeger and Kohler, 1976; Majumder, 1981; Halder and Majumder, 1986; Dey and Majumder, 1990; Walter et al, 2000; Guthmann et al, 2002; for review Nath et al, 2008), the above‐mentioned findings from our laboratory provide confirmatory evidence for the localization of an ecto‐protein kinase and its protein substrate on a cell surface.…”

Ecto-Cyclic AMP Independent Protein Kinase-A Potent Regulator of L-type Calcium Channel and Forward Motility of Goat (Capra indicus) Epididymal Spermatozoa

“…Thereby, it demonstrates that CIK is a unique membrane protein-specific kinase, which specifically phosphorylates serine and threonine residues of the sperm outer cellsurface phosphoproteins [35]. It was further demonstrated that ecto-CIK plays a vital role in the regulation of sperm forward progression and velocity through its substrate protein (MPS) [33][34][35][36]. The aim of this present study is to investigate the role of the phosphorylation event mediated by the CIK on the activation of forward motility in the epididymis through plasma membrane bound voltage dependent L type calcium channel.…”

A Novel Membrane Protein-Specific Serine/Threonine Kinase: Tissue Distribution and Role in Sperm Maturation

“…Both CIK and MPS are localized primarily in the acrosomal cap area of the external surface of the mature sperm head as demonstrated by indirect immunofluorescence studies [4, 6]. We have demonstrated that ecto-CIK through its substrate protein (MPS) plays a vital role in the regulation of sperm forward progression and velocity [4, 5, 7]. More recent studies from our laboratory demonstrated that the sperm external surface CIK and MPS are essential for membrane fusion component of acrosome reaction [6].…”