Calcitriol induces post-thawed bovine sperm capacitation

Background:Capacitation is a set of physiological changes sperms undergo to acquire fertilizing capacity.In vivo,this process is directly associated with high calcium levels in sperm cytoplasm.Calcitriol,the vitamin D hypercalcemic metabolite,is related to human sperm motility,capacitation,and acrosome reaction.This work aimed to study the effect of calcitriol on bull sperm quality parameters and capacitation.Methods:One million freezethawed spermatozoa were obtained from different bulls and treated with 20 nM of calcitriol for 30 min.Untreated cells(negative control)and treated ones with calcitriol or heparin(100μg/mL,positive capacitation control)were evaluated for motility,viability,and functional parameters.Menadione(70μM,30 min)treatment was included as a reactive oxygen species(ROS)positive sperm agent.Results:The results elucidated that sperm exposed to 20 nM calcitriol showed higher viability,vigor,and capacitation than their positive and negative controls.The percentage of sperm with intact plasma and acrosome membranes,mitochondrial membrane potential(ΔΨm),and phosphatidylserine externalization was similar in all the conditions evaluated,while ROS production was higher with heparin and menadione-treated groups than the calcitriol group or negative control.Conclusion:Our results indicate that calcitriol induces the capacitation of thawed bull spermatozoa and maintains acceptable values of progressive motility,viability,and vigor without altering key biological parameters such as redox status,ΔΨm,and cell death.

Mechanism of sperm capacitation and the acrosome reaction： role of protein kinases

Mammalian sperm must undergo a series of biochemical and physiological modifications, collectively called capacitation, in the female reproductive tract prior to the acrosome reaction （AR）. The mechanisms of these modifications are not well characterized though protein kinases were shown to be involved in the regulation of intracellular Ca2＋ during both capacitation and the AR. In the present review, we summarize some of the signaling events that are involved in capacitation. During the capacitation process, phosphatidyl-inositol-3-kinase （PI3K） is phosphorylated/activated via a protein kinase A （PKA）-dependent cascade, and downregulated by protein kinase C a （PKCa）. PKCa is active at the beginning of capacitation, resulting in PI3K inactivation. During capacitation, PKCa as well as PP172 is degraded by a PKA-dependent mechanism, allowing the activation of PI3K. The activation of PKA during capacitation depends mainly on cyclic adenosine monophosphate （cAMP） produced by the bicarbonate-dependent soluble adenylyl cyclase. This activation of PKA leads to an increase in actin polymerization, an essential process for the development of hyperactivated motility, which is necessary for successful fertilization. Actin polymerization is mediated by PIP2 in two ways： first, PIP2 acts as a cofactor for phospholipase D （PLD） activation, and second, as a molecule that binds and inhibits actin-severing proteins such as gelsolin. Tyrosine phosphorylation of gelsolin during capacitation by Src family kinase （SFK） is also important for its inactivation. Prior to the AR, gelsolin is released from PIP2 and undergoes dephosphorylation/activation, resulting in fast F-actin depolymerization, leading to the AR.

The role of ezrin-associated protein network in human sperm capacitation

Membrane modifications in sperm cells represent a key step in sperm capacitation; however, the molecular basis of these modifications is not fully understood. Ezrin is the best-studied member of the ezrin/radixin/merlin family. As a cross-linker between the cortical cytoskeleton and plasma membrane proteins, ezrin contributes to remodeling of the membrane surface structure. Furthermore, activated ezrin and the Rho dissociation inhibitor, RhoGDI, promote the formation of cortical cytoskeleton-polymerized actin through Rho activation. Thus, ezrin, actin, RhoGDI, Rho and plasma membrane proteins form a complicated network in vivo, which contributes to the assembly of the structure of the membrane surface. Previously, we showed that ezrin and RhoGDI1 are expressed in human testes. Thus, we sought to determine whether the ezrin-RhoGDIl-actin-membrane protein network has a role in human sperm capacitation. Our results by Western blot indicate that ezrin is activated by phosphorylation of the threonine567 residue during capacitation. Co-immunoprecipitation studies revealed that, during sperm capacitation, the interaction between ezrin and RhoGDI1 increases, and phosphostaining of two dimensional electrophoresis gels showed that RhoGDI 1 is phosphorylated, suggesting that RhoGDI 1 dissociates from RhoA and leads to actin polymerization on the sperm head. We speculate that activated ezrin interacts with polymerized actin and the glycosylated membrane protein cd44 after capacitation. Blocking sperm capacitation using ezrin- or actin-specific monoclonal antibodies decreases their acrosome reaction （AR） rate, but has no effect on the AR alone. Taken together, our results show that a network consisting of ezrin, RhoGDI1, RhoA, F-actin and membrane proteins functions to influence the modifications that occur on the membrane of the sperm head during human sperm capacitation.

Carbohydrates mediate sperm-ovum adhesion and triggering of the acrosome reaction

The fertilization process is the net result of a complex sequence of events that collectively result in the fusion of theopposite gametes. The male gamete undergoes continuous morphological and biochemical modifications during spermdevelopment in the testis (spermatogenesis), maturation in the epididymis, and capacitation in the female reproductivetract. Only the capacitated spermatozoa are able to recognize and bind to the bioactive glycan residue(s) on the ovum'sextracellular coat, the zona pellucida (ZP). Sperm-zona binding in the mouse and several other species is believed totake place in two stages. First, capacitated (acrosome-intact) spermatozoa loosely and reversibly adhere to the zona-in-tact ovum. In the second stage tight irreversible binding occurs. Both types of bindings are attributed to the presence ofglycan- binding proteins (receptors) on the sperm plasma membrane and their complementary bioactive glycan units(ligands) on the surface of the ZP. The carbohydrate-mediated adhesion event initiates a signal transduction cascade re-sulting in the exocytosis of acrosomal contents. This step is believed to be prerequisite which allows the hyperactivatedacrosome-reacted spermatozoa to penetrate the ZP and fertilize the ovum. This review focuses on the role of carbohy-drate residues in sperm-ovum interaction, and triggering of the acrosome reaction. I have attempted to discuss extensiveprogress that has been made to enhance our understanding of the well programmed multiple molecular events necessaryfor successful fertilization. This review will identify these events, and discuss the functional significance of carbohy-drates in these events.

Insulin affects sperm capacity in pig through nitric oxide

Insulin （Ins） has recently been demonstrated to have the ability to induce the capacitation process in pig spermatozoa. In various mammalian species, capacitation has been linked to the nitric oxide （NO） signalling; therefore, this study investigated NO production in Ins-treated pig spermatozoa by fluorescence-activated cell sorting. For the same samples, sperm capacitation was evaluated chlortetracycline staining, protein tyrosine phosphorylation pattern and acrosomal status. A significant increase of the intrasperm NO level and the activation of three capacitation indices were detected in response to Ins treatment. Conversely, sperm preincubation with an NO synthase inhibitor （N-nitro-L-arginine methyl ester） or with the anti-Ins receptor β （IRβ） antibody reversed all of the Ins-related effects. These results suggest that Ins has the capacity to enhance intracellular NO concentrations in pig spermatozoa and indicate a oossible NO implication upon Ins oromotion of caoacitation.

Decreased AKAP4/PKA signaling pathway in high DFI sperm affects sperm capacitation

The sperm DNA fragmentation index(DFI)is a metric used to assess DNA fragmentation within sperm.During in vitro fertilizationembryo transfer(IVF-ET),high sperm DFI can lead to a low fertilization rate,poor embryo development,early miscarriage,etc.A kinase anchoring protein(AKAP)is a scaffold protein that can bind protein kinase A(PKA)to subcellular sites of specific substrates and protects the biophosphorylation reaction.Sperm protein antigen 17(SPA17)can also bind to AKAP.This study intends to explore the reason for the decreased fertilization rate observed in high sperm DFI(H-DFI)patients during IVF-ET.In addition,the study investigates the expression of AKAP,protein kinase A regulatory subunit(PKARIl),and SPA17 between H-DFI and low sperm DFI(L-DFI)patients.SPA17 at the transcriptional level is abnormal,the translational level increases in H-DFI patients,and the expression of AKAP4/PKARIl protein decreases.H,O,has been used to simulate oxidative stress damage to spermatozoa during the formation of sperm DFI.It indicates that H,O,increases the expression of sperm SPA17 protein and suppresses AKAP4/PKARIl protein expression.These processes inhibit sperm capacitation and reduce acrosomal reactions.Embryo culture data and IVF outcomes have been documented.The H-DFI group has a lower fertilization rate.Therefore,the results indicate that the possible causes for the decreased fertilization rate in the H-DFI patients have included loss of sperm AKAP4/PKARIl proteins,blocked sperm capacitation,and reduced occurrence of acrosome reaction.

Molecular aspects of mammalian fertilization

Mammalian fertilization is a highly regulated process, much of which are not clearly understood. Here we presentsome information in order to elaborate a working hypothesis for this process, beginning with the sperm modifications inthe epidydimis up to sperm and egg plasmalemma interaction and fusion. We also discuss the still poorly understood ca-pacitation process, the phenomenon of sperm chemo-attraction that brings the capacitated sperm to interact with theoocyte vestments and certain aspects of the acrosome reaction. (Asian J Androl 2001 Dec; 3 : 243 - 249)

Are sperm capacitation and apoptosis the opposite ends of a continuum driven by oxidative stress？

This chapter explores the possibility that capacitation and apoptosis are linked processes joined by their common dependence on the continued generation of reactive oxygen species （ROS）. According to this model capacitation is initiated in spematozoa following their release into the female reproductive tract as a consequence of intracellular ROS generation, which stimulates intracellular cAMP generation, inhibits tyrosine phosphatase activity and enhances the formation of oxysterols prior to their removal from the sperm surface by albumin. The continued generation of ROS by capacitating populations of spermatozoa eventually overwhelms the limited capacity of these cells to protect themselves from oxidative stress. As a result the over-capacitation of spermatozoa leads to a state of senescence and the activation of a truncated intrinsic apoptotic cascade characterized by enhanced mitochondrial RO$ generation, lipid peroxidation, motility loss, caspase activation and phosphatidylserine externalization. The latter may be particularly important in instructing phagocytic leukocytes that the removal of senescent, moribund spermatozoa should be a silent process unaccompanied by the generation of proinflammatory cytokines. These observations reveal the central role played by redox chemistry in defining the life and death of spermatozoa. A knowledge of these mechanisms may help us to engineer novel solutions to both support and preserve the functionality of these highly specialized cells.

An epididymis-specific carboxyl esterase CES5A is required for sperm capacitation and male fertility in the rat

Despite the fact that the phenomenon of capacitation was discovered over half century ago and much progress has been made in identifying sperm events involved in capacitation, few specific molecules of epididymal origin have been identified as being directly involved in this process in vivo. Previously, our group cloned and characterized a carboxyl esterase gene CesSa in the rat epididymis. The CES5A protein is mainly expressed in the corpus and cauda epididymidis and secreted into the corresponding lumens. Here, we report the function of CESSA in sperm maturation. By local injection of Lentivirus-mediated siRNA in the CESSA.expressing region of the rat epididymis, CesSa-knockdown animal models were created. These animals exhibited an inhibited sperm capacitation and a reduction in male fertility. These results suggest that CESSA plays an important role in sperm maturation and male fertility.

Remodeling of the plasma for sperm-egg recognition： proteins membrane in preparation roles of acrosomal

The interaction of sperm with the egg＇s extracellular matrix, the zona pellucida （ZP） is the first step of the union between male and female gametes. The molecular mechanisms of this process have been studied for the past six decades with the results obtained being both interesting and confusing. In this article, we describe our recent work, which attempts to address two lines of questions from previous studies. First, because there are numerous ZP binding proteins reported byvarious researchers, how do these proteins act together in sperm-ZP interaction？ Second, why do a number of acrosomal proteins have ZP affinity？ Are they involved mainly in the initial sperm-ZP binding or rather in anchoring acrosome reacting/reacted spermatozoa to the ZP？ Our studies reveal that a number of ZP binding proteins and chaperones, extracted from the anterior sperm head plasma membrane, coexist as high molecular weight （HMW） complexes, and that these complexes in capacitated spermatozoa have preferential ability to bind to the ZP. Zonadhesin （ZAN）, known as an acrosomal protein with ZP affinity, is one of these proteins in the HMW complexes. Immunoprecipitation indicates that ZAN interacts with other acrosomal proteins, proacrosin/acrosin and sp32 （ACRBP）, also present in the HMW complexes. Immunodetection of ZAN and proacrosin/acrosin on spermatozoa further indicates that both proteins traffic to the sperm head surface during capacitation where the sperm acrosomal matrix is still intact, and therefore they are likely involved in the initial sperm-ZP binding step.