Spermatozoa recruit prostasomes in response to capacitation induction

Abstract

Seminal plasma contains various types of extracellular vesicles, including ‘prostasomes’. Prostasomes are small vesicles secreted by prostatic epithelial cells that can be recruited by and fuse with sperm cells in response of progesterone that is released by oocyte surrounding cumulus cells. This delivers Ca^2 + signaling tools that allow the sperm cell to gain hypermotility and undergo the acrosome reaction. Conditions for binding of prostasomes to sperm cells are however unclear. We found that classically used prostasome markers are in fact heterogeneously expressed on distinct populations of small and large vesicles in seminal plasma. To study interactions between prostasomes and spermatozoa we used the stallion as a model organism. A homogeneous population of ~ 60 nm prostasomes was first separated from larger vesicles and labeled with biotin. Binding of biotinylated prostasomes to individual live spermatozoa was then monitored by flow cytometry. Contrary to assumptions in the literature, we found that such highly purified prostasomes bound to live sperm only after capacitation had been initiated, and specifically at pH ≥ 7.5. Using fluorescence microscopy, we observed that prostasomes bound primarily to the head of live sperm. We propose that in vivo, prostasomes may bind to sperm cells in the uterus, to be carried in association with sperm cells into oviduct and to fuse with the sperm cell only during the final approach of the oocyte. This article is part of a Special Issue entitled: An Updated Secretome.

Introduction

During or directly after ejaculation, sperm cells are mixed with secretions from the prostate and other accessory sex glands (i.e. seminal vesicles, bulbo-urethral glands). Prostatic fluid contains a high concentration of citrate, which serves as an energy source for sperm [1], and enzymes that function in the liquefaction of coagulated semen, including prostate-specific antigen and prostatic acid phosphatase [2], [3]. In addition to soluble constituents, seminal fluid from many mammalian species, including man, the pig, cow, sheep and horse has been found to contain various types of extracellular vesicles, including prostasomes [4], [5], [6], [7], [8], [9], [10]. Prostasomes are generated within prostate epithelial cells as 30–130 nm intraluminal vesicles contained within multivesicular bodies [11]; subsequent fusion of the limiting membrane of these multivesicular bodies with the cell plasma membrane results in the secretion of the contained intraluminal vesicles, i.e. the prostasomes, into the extracellular prostatic fluid [9], [12] in a process similar to the production of exosomes by other cell types [13]. Although membrane vesicles in seminal plasma may originate from various organs within the male reproductive tract [14], the prostate is considered to be the major source of small membrane vesicles in both man [15] and the stallion [16].

Prostasomes have a characteristic molecular composition. Regarding their lipid content, their fatty acids are mostly saturated or monounsaturated, and unusual high concentrations of cholesterol and sphingomyelin are present [17], [18]. The most extensively studied prostasomal proteins are enzymes that had already been identified before these vesicles had been analyzed using modern proteomic techniques. These include dipeptidyl peptidase IV (DPP4; CD26) [19], [20], aminopeptidase N [20], 5′nucleotidase [21], alkaline phosphatase, alkaline phosphodiesterase I [22], neutral endopeptidase [23], type 2 transmembrane serine protease (TMPRSS2) [24], ecto-diadenosine polyphosphates hydrolase [25], PKA and PKC, casein kinase II, and membrane-bound ATPase [26]. Comprehensive proteomic analyses of human prostasomes have also been performed. In a first study, 139 proteins were identified, which besides enzymes included transport and structural proteins, GTP-binding and other signaling proteins, chaperones, and non-annotated proteins [27]. In a later study as many as 440 proteins were identified [28]. Prostasome markers that are of particular interest are the prostate specific proteins prostatic acid phosphatase (PAP), prostate specific antigen (PSA), TMPRSS2 [27], prostate specific transglutaminase [27], [28] and prostate stem cell antigen (PSCA) [28]. Some prostasomal proteins have already been studied as (candidate) markers for prostate cancer [2], [29], [30], [31], of which PSA is now well established in the clinic. It should be noted that prostasome isolates can be contaminated with membranes originating from tissues other than the prostate, and that different subclasses of prostasomes with unique protein components have been characterized [10]. Therefore, the definite proteome of prostasomes remains to be established.

The proposed functions of prostasomes include prevention of immune-mediated recognition or destruction of spermatozoa within the female reproductive tract [32], [33], [34], [35], [36], [37]. An alternative or additional function lies in their apparent capacity to modulate the activation status of sperm cells. After release into the female reproductive tract, mammalian sperm cells first need to acquire fertilizing potential via ‘capacitation’, a complex series of processes that encompass an increase in intracellular concentrations of reactive oxygen species, Ca^2 + and cAMP, protein tyrosine phosphorylation, and rearrangement of plasma membrane proteins and lipids (reviewed in [38], [39]). Capacitation is initiated in response to the favorable physiological environment within the female reproductive tract, with important elements of this environment including the relatively high concentrations of cholesterol binding proteins/complexes, Ca^2 + and HCO₃⁻. Sperm cells contain a special soluble adenylyl cyclase as well as several membrane associated adenylyl cyclases. The soluble adenylyl cyclase is mainly expressed in the testis, especially present in spermatozoa, and indispensable for fertility, as demonstrated for soluble adenylyl cyclase knock-out mice [40]. Early capacitation events include the activation of soluble adenylyl cyclase by HCO₃⁻, resulting in cAMP induced protein kinase A (PKA)-dependent protein phosphorylation [41]. This leads to a rearrangement in the plasma membrane lipid asymmetry and lateral organization [42], which is thought to facilitate the extraction of cholesterol from membranes [43] and to contribute in changing the activities of relevant G protein-coupled receptors and membrane associated adenylyl cyclases [39], [44]. In this way both soluble and membrane-associated adenylyl cyclases indirectly regulate tyrosine phosphorylation of a number of proteins later during capacitation.

Capacitated sperm alter their motility characteristics, in a manner thought to facilitate their passage through the latter parts of the female reproductive tract [45], and are primed to undergo the acrosome reaction in case they should contact the zona pellucida and/or cumulus cells surrounding the oocyte [46]. Cells that acrosome-react before contacting these structures are incapable of fertilizing, because local release of the acrosomal enzymes is essential for successful penetration of the zona pellucida. Since only a few sperm cells manage to reach the oocyte in vivo [47], premature spontaneous acrosome reaction by capacitated spermatozoa needs to be prevented. In this respect, seminal fluid appears to contain several factors capable of inhibiting capacitation (reviewed by [39]). For example, prostasomes have a relatively high cholesterol content [15] and have therefore been proposed to inhibit membrane reorganization during capacitation and the subsequent acrosome reaction by donating cholesterol to the sperm plasma membrane [48], [49], although opposing studies have suggested that prostasomes actually stimulate the acrosome reaction [50], [51]. Another function of prostasomes was indicated by the recent demonstration that they contain and can transfer the Ca^2 +-signaling tools required for sperm cell hyperactive motility, a required characteristic for zona pellucida penetration [52]. Membrane fusion of prostasomes with sperm cells can be initiated in response of progesterone that is released by oocyte surrounding cumulus cells. This poses a conceptual problem: When prostasomes fuse with sperm cells only while approaching the oocyte, how do they get into the oviduct?

For studies on prostasome function, the horse is an attractive model because equine semen can easily be obtained in large quantities, and the characteristics of equine prostasomes appear to be very similar to those from men [8]. To resolve questions regarding the requirements for prostasome binding to sperm, we analyzed in vitro binding of highly purified, labeled prostasomes to spermatozoa, while distinguishing live from dead cells and capacitated from non-capacitated cells. Our observations may in part explain controversies in the literature regarding the conditions for prostasome binding and the potential role of prostasomes in sperm capacitation and the acrosome reaction [48], [49], [50], [51].