Recombinant human ZP3-induced sperm acrosome reaction: Evidence for the involvement of T- and L-type voltage-gated calcium channels

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Abstract

For successful fertilization mammalian spermatozoa must undergo the acrosome reaction (AR), an exocytotic event that allows this cell to penetrate the outer layer of the oocyte, the zona pellucida (ZP). Four glycoproteins (ZP1–ZP4) compose the human ZP, being ZP3 the physiological inductor of the AR. This process requires changes in intracellular Ca2+ concentration ([Ca2+]i) involving not fully understood mechanisms. Even in mouse sperm, the pharmacologically documented participation of voltage-gated Ca2+ (CaV) channels and store-operated channels (SOCs) in the ZP-induced AR is being debated. The situation in human sperm is even less clear due to the limited availability of human ZP. Here, we used recombinant human ZP3 (rhZP3) produced in baculovirus-infected Sf9 cells to investigate the involvement of CaV channels in the human sperm AR. Our findings showed that Ni2+ and mibefradil at concentrations that block T-type or CaV3 channels, and nimodipine and diltiazem that block L-type or CaV1 channels, significantly inhibited the rhZP3-initiated AR. On the other hand, the AR was insensitive to concentrations of ω-Agatoxin IVA, ω-Conotoxin GVIA and SNX-482 that block P/Q, N and R-type channels, respectively (CaV2 channels). Our overall findings suggest that CaV1 and CaV3 channels participate in human sperm AR. Consistent with this, we detected in human sperm transcripts for the CaV1 auxiliary subunits, α2δ, β1, β2 and β4, but not the neuronal specific isoforms β3 and γ2.

Introduction

The influx of extracellular Ca2+ into sperm through specific channels in the plasma membrane is a necessary early event for acrosome reaction (AR) in mammals. Immunolocalization and pharmacological approaches have suggested the involvement of voltage-gated Ca2+ (CaV) channels during the AR [1]. The CaV channel family consists of three subfamilies: CaV1 and CaV2 encode high voltage activated channels (HVA) constituted by seven different genes coding for L, P/Q, N and R currents, CaV3 encode low voltage activated channels (LVA, or T-type Ca2+ channels) with three different genes. Much of our current understanding of the functional roles played by CaV channels in the AR has been obtained using the mouse as a model system [1]. Less is known about the human sperm AR and growing evidence indicates that there are significant differences between these two species.

Previous studies have provided strong evidence indicating that the egg zona pellucida (ZP) is the physiological inducer of sperm AR [2]. The ZP is composed of a matrix of glycoproteins surrounding the egg. While mouse ZP contains three glycoproteins, human ZP contains four (ZP1–ZP4) and the glycosylation pattern of these proteins seems to be important for their biological activity [2]. In particular, ZP3 induces a [Ca2+]i signal that triggers mouse sperm AR. Its initial component is a transient [Ca2+]i increase with kinetics and pharmacology consistent to those of T-currents from mouse spermatogenic cells and the sperm AR [3]. This initial Ca2+ entry is followed by a second sustained Ca2+ influx mediated primarily by store-operated channels (SOCs) [1].

Progress in elucidating the molecular mechanisms involved in human sperm AR have been greatly halted by the scarcity of biological material available for experimental research. Therefore, diverse substances including progesterone (P4) have been used to induce the human sperm AR. Micromolar concentrations of P4 induce a transient elevation of sperm [Ca2+]i that reflects an influx of extracellular Ca2+, which is sensitive to inhibitors of CaV channels [4]. After the initial response, a second, sustained elevation of [Ca2+]i occurs and the AR is induced. Instead, exposure of sperm to an increasing P4 gradient induces a slow [Ca2+]i rise with superimposed oscillations. These oscillations do not induce the AR, but modulate flagellar activity promoting motility that persists even in the absence of Ca2+, suggesting mobilization of this ion from internal stores [4].

In contrast, there is limited information about the ZP-induced AR in human sperm. A few studies have been performed either using ZP proteins isolated from human eggs obtained post mortem, or ZP purified from discarded oocytes originally destined for in vitro fertilization procedures [5]. Studies with individually purified ZP proteins are even scarcer, in particular with ZP4, which does not have a counterpart in mouse sperm. Recently, the participation of CaV1 and CaV3 channels in response to purified native ZP3 and ZP4 from remnants of in vitro fertilization oocytes was reported in the human sperm AR [6]. However, in that study only a limited pharmacological analysis was done due to the restricted availability of ZP3 and ZP4. Although the glycosylation profile of recombinant human ZP proteins is not identical to that of the native proteins, they are an alternative tool to study the ZP-induced AR [7], [8]. Recombinant proteins may help to unravel the involvement of CaV channels in the ZP-induced AR in human sperm. Herein, we have used recombinant human ZP3 (rhZP3) expressed in baculovirus-infected Sf9 cells to characterize the CaV channels involved in the AR of human capacitated sperm. We also report for the first time that L-type auxiliary subunits are expressed in human sperm.

Section snippets

Materials

Bovine serum albumin (BSA), A23187, Ham’s F-10 media, fluorescein isothiocyanate-Pisum sativum agglutinin (FITC-PSA), diltiazem, verapamil and SNX-482 were purchased from Sigma–Aldrich Chemical Co. (St. Louis, MO). Nimodipine, calciseptine, ω-Conotoxin GVIA and nifedipine were from Alomone Labs (Jerusalem, Israel). ω-Agatoxin IVA was from EMD Chemicals Inc. (Darmstadt, Germany). Mibefradil and human brain cDNA were generous gifts from Drs. J.C. Gomora (IFC, UNAM, México) and R. Gutiérrez

Results

Initially, we tested the ability of the purified rhZP2, ZP3 and ZP4 to induce human sperm AR. Both rhZP3 (10 ng/μl) or rhZP4 (10 ng/μl) were able to significantly induce AR in capacitated sperm, compared with control condition (Fig. 1). Unexpectedly, although to a lesser extent, both proteins also induced AR in non-capacitated sperm. As anticipated, there was no significant induction of AR in capacitated or non-capacitated sperm with rhZP2 (10 ng/μl). The Ca2+ ionophore A23187 (10 μM) was used as a

Discussion

The scarcity of native ZP has halted the understanding of physiologically induced AR. Our findings illustrate the pertinence of using recombinant proteins. The quantities and rapid production of these proteins allow the performance of a variety of studies and pharmacological screenings.

As mentioned earlier, CaV channels appear to play a major role in mouse sperm. The CaV3 subfamily is composed by CaV3.1, CaV3.2 and CaV3.3 [19] subunits. In particular, CaV3.2 has been described as essential for

Role of the funding source

This work was supported financially by NIH Grants R01 HD03808207A1 (to A.D.), CONACyT-Mexico (49113 to A.D., 39860 to R.F., 47011 to M.C. and 99333 to C.T.), DGAPA/UNAM (IN211809 to A.D. and IN204109 to C.T.).

Acknowledgments

The authors thank Shirley Ainsworth, Marcela Ramírez, Juan Manuel Hurtado, Roberto Rodríguez and Elizabeth Mata for technical assistance.

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