Molecular aspects of mammalian fertilization

Hector Serrano¹,  Dolores Garcia-Suarez²

¹Dept. Health Sciences, ²Dept. Biology, Universidad Autonma Metropolitana-Iztapalapa, Ave. Michoacan and Purisima, Mexico City, DF 09340, Mexico

Asian J Androl  2001 Dec; 3: 243-249

Keywords:  fertilization; sperm maturation; sperm capacitation; sperm-ovum interactions; membrane fusion

Abstract

Mammalian fertilization is a highly regulated process, much of which are not clearly  understood. Here we present some information in order to elaborate a working hypothesis for this process, beginning with the sperm modifications in the epidydimis up to sperm and egg plasmalemma interaction and fusion. We also discuss the still poorly understood capacitation process, the phenomenon of sperm chemo-attraction that brings the capacitated sperm to interact with the oocyte vestments and certain aspects of the acrosome reaction.

1 Introduction

Fertilization is a process that is the end of one developmental process and the beginning of another process. On the one side, it is the end of the oogenesis and spermatogenesis processes. On the other side, it is the starting point of a new organism. Several aspects of the fertilization process have been described by using different techniques ranging from simple microscopical analysis to knock out genes and transgenic animals. Much of our actual knowledge at the molecular level has been developed in the last two decades but regardless the large amountresearch effort, there are some aspects that are still poorly understood. In this review, we are not trying to introduce all known information of mammalian fertilization that has been covered by other authors^[1,2],  but to assemble some described models for different topics of the process from the spermatozoa perspective, in order to make them understandable and to detect the still uncovered areas in each model.

2 Sperm maturation and epididymis

The glycoproteins on the sperm plasma membrane are different in origin. Galactosyltransferase, an enzyme participating in gamete interaction in the rat, is located on the plasma membrane of spermatocytes and the postacrosomal region of tubular spermatozoa and later on the acrosome region of the sperm recovered from the female genital tract^[3]. Other proteins are not produced by the spermatocytes but are secreted by the epithelial cells lining the epididymis, like the DE protein in the rat^[4-6], or by the male accessory glands or the female oviduct, like the oviduct-specific glycoprotein (OSP) that is important in both fertilization and early embryonic development^[7].  The secretory and absorptive activities of the epithelia lining the epididymis are responsible for the surface sculpturing of the mammalian sperm. Gene expression along the epididymis actively participates in this change in shape and characteristics of the mammalian spermatozoa^[8].

The relatively diverse origin of the proteins actually lead to the establishment of sperm membrane domains responsible for different sperm capabilities during the fertilization process. Some molecules fixed on the sperm membrane during spermiogenesis can be processed later in the prostate or the female genital tract in such a way that this new conformation can acquire a function that otherwise can not be achieved, as is the case of fibronectin, which is exposed on the human sperm surface only after capacitation has occurred^[9]. 

3 Capacitation and transport in the female genital tract

When sperm are deposited into the female genital tract, they interact with different compounds. Some of these compounds are in the prostatic secretion. Some others, like mucins, are secreted at various parts of the female genital tract and some of them actually combines with those in the prostatic secretions present in ejaculates, composing the first barrier that the sperm will be gradually released.

In most eutheria, even when several million sperm are deposited in the initial parts of the female tract, only a small part are able to be liberated from the initial barrier. Some species like bull, cattle, pigs, rabbits, mouse and hamsters have anatomically or functionally specialized sperm reservoirs^[14-16]. In bulls, recent studies have demonstrated that modification or loss of a sperm membrane peripheral protein acquired from the seminal plasma^[14] through  interacting with the fructose residues on the epithelial cell membrane is responsible for sperm liberation from the functional storage at different times^[16]. The liberated sperm then continue their journey up to the fertilization site^[15].

Once the spermatozoa have been released, they are subjected to different physiological processes. One of these is capacitation, which is initiated almost at the same time the sperm is ejaculated. During the sperm-epithelial cells interaction, there are lipid exchange and proteolytic and carbohydrate modification of the sperm surface. Evidences for most of the processes are derived from the requirements of spermatozoa in order to successfully interact with the oocyte^[17].

During capacitation, the sperm undergo several characteristic biochemical and morphological changes. Chlortetracycline staining^[18], for example, is able to demonstrate the capacitation status as well as to evaluate the acrosome reaction^[19].

Albumin is a characteristic component of in vitro capacitation media that presumably removes the cholesterol from the sperm plasma membrane, thus increasing the membrane fluidity^[20]. Therien and co-workers^[21] recently proposed that in regard to lipid exchange, bull sperm are subjected to two cholesterol effluxes. One occurs after a short interaction with the seminal plasma where the bovine seminal plasma proteins (BSP) remove a significantly large amount of cholesterol and a small amount of other phospholipids that initially destabilizes (primes) the membrane. These BSP proteins remain attached to the sperm plasma membrane through their interactions with choline phospholipids that prevents further phospholipid movement. In the female genital tract, a second protein, high density lipoprotein from the follicular fluid (FF-HDL), removes the BSP proteins from the plasmalema so the cholesterol and other phospholipids can be removed by low affinity proteins like albumin.

During capacitation, there are changes on the sperm surface related to the modification or releasing of molecules due to the action of modifying or hydrolyzing enzymes in such a way that new antigenic determinants are detected. From the time-based definition of capacitation^[22] to its biochemical-physiological aspects^[19], our knowledge on capacitation is far from complete.

Calmodulin plays a major role in the capacitation as the process is dependent on extracellular Ca²⁺. In mouse, calmodulin possibly activates adenylyl cyclase but inhibition of calmodulin does not affect the tyrosine phosphorylation characteristic of capacitation^[23]. This controversial result may be explained by the following observation. cAMP is necessary for the activation of certain kinases, but it can effectively act at low concentration that is not affected by calmodulin-cyclase interaction. Calmodulin inhibition also impairs other capacitation indicator. Calmodulin plays an important role in the whiplash bending of the sperm tail known as hyperactivation, which is not cAMP-dependent^[23].

In hamster sperm, capacitation-associated changes depend mainly on the bicarbonate-specific transporter associated to the cAMP metabolism. Capacitation is also related to protein phosphorylation by the cAMP-dependent protein kinase (PKA) and some other pathway, however, this kind of activation is not enough to obtain fully capacitated spermatozoa.

Reactive oxygen species (ROS) do regulate oxidation-reduction processes needed for protein phosphorylation. Presence of nitric oxide (NO) affects the most obvious signals of capacitation, i.e., hyperactivated motility and egg-envelope interaction^[24]. Recently it has been demonstrated that NO correlates with the protein phosphorylation level in human spermatozoa. It has been proposed that NO and cAMP interaction has a stimulatory effect on specific tyrosine-phosphorylation.

4 Sperm maintaining and attracting substances

In the female genital tract spermatozoa has to be maintained by different energetic sources. Vaginal, tubal and follicular secretions modify not only sperm surface characteristics but also their motility and responsiveness. In vitro assays have tried to emulate some of the different environments the sperm found along its travel to the fertilization site^[26].

5 Acrosome reaction

Acrosome reaction is a highly specialized event observed in the capacitated spermatozoa.  Although it has been extensively studied and there are several techniques available for its evaluation^[18,41-43], the molecular events that trigger the acrosome reaction have not been fully elucidated. Gamete interaction is the induction signal for this exocytic event and it is admitted that the natural acrosome reaction inducer is the zona pellucida. In vitro studies also indicate that factors from the follicular fluid, mainly progesterone(P₄), are able to induce the reaction. At least two stimulatory transduction pathways have been implicated.

Let's start with the P4-induced acrosome reaction. After the sperm has traveled up to the isthmus, the interaction that induces sperm membrane changes modifies the structure of previously unavailable determinants to interact. Progesterone interaction with sperm is different from that usually shown in other P₄-responsive cells. By using fluorescence-labeled P₄ probes, several groups have found a specific receptor located on the plasmalemma of different sperm species from mouse to pigs and bulls^[44].

Membrane progesterone receptor is not a G-protein coupled receptor. P4-receptor interaction increases the intracellular Ca²⁺ concentration as well as the long lasting increase in Na⁺ concentration due to a flunarizine-sensitive channel^[45].

Progesterone stimulation has been revealed in mouse sperm by an intracellular Ca²⁺ increase. There is controversial evidence about the type of Ca²⁺ channel involved. Some authors suggest that the rise in Ca²⁺ concentration is not due to the action of the T-type Ca²⁺ channel but another one also activated by cGMP^[46], whereas others suggest that there are other channels acting during the zona pellucida-induced acrosome reaction^[45,47]. The P₄ receptor acts first via a cAMP-dependent protein kinase (PKA) and then by an A-kinase anchored protein (AKAP).

This system mediates the cytoplasmic transportation of the PKA active fragment that in other cells interacts with some other cytoplasmic compartments or even with specific molecules such as those involved in Ca²⁺ regulation: calmodulin, calreticulin and calbindin^[48].

If this model also can be applied to the spermatozoa, it is reasonable to postulate that after progesterone interaction with the membrane receptor, the activation of the adenylyl cyclase increases cAMP concentration. This second messenger then permits the activation of the PKA-AKAP system in such a way that the regulatory moiety of the PKA can interact with both calmodulin and some type of Ca²⁺channel, thus increasing the intracellular Ca²⁺ concentration. This increase then promotes a change in the cytoskeletal organization in such a way that, the zone comprising the outer acrosomal membrane and the plasma membrane covering the acrosome will fuse at several points and release the acrosome content.

Membrane fusion is also a time-dependent event that at the early stages only small membrane regions are involved and later more will be included^[1].Leyton and Saling^[49], after partial proteolysis of mouse zona pellucida demonstrated that some small peptides could interact with capacitated, but acrosome intact mouse spermatozoa. These peptides do not induce the acrosome reaction until the zona receptors are aggregated by a bivalent antibody. Carrera and co-workers^[50] indicate that incubation of capacitated sperm with zona pellucida promotes a trans-phosphorylation process after aggregation of a tyrosine-kinase type receptor. These phosphorylated receptors interact with kinase-type proteins that could explain the increase in phosphorylated proteins found during the acrosome reaction^[19,51].

The pivotal role of Ca²⁺ in the acrosome reaction has been demonstrated by a wide variety of techniques, as the Ca²⁺ specific ionosphere and the concentration-sensitive fluorescent probes. To explain the source of Ca²⁺, some authors have demonstrated that the most important source is the surrounding medium. Acrosome reaction can be observed in sperm incubated in a Ca²⁺ containing medium, but not in Ca²⁺ free medium. Polyamines, particularly spermidine, are required for a successful Ca²⁺ rise^[52]. If the Ca²⁺ source is intracellular, the main source should be the mitochondria. In spermatozoa, Ca²⁺ regulating proteins other than calmodulin have not been demonstrated.

6 Sperm-egg interaction, zona pellucida penetration and membrane fusion

Recently, several reports using lectins or specific antibodies have characterized some molecules involved in gamete interaction leading to gamete fusion. In the mice, the O-linked carbohydrates and the mouse ZP₃ protein core^[54], whereas in the pigs, the tri- and tetra- antennary neutral N-linked oligosaccharides^[55], a lactosaminoglycan moiety located as discrete layers through the zona pellucida, and three glycoproteins^[56], are responsible for sperm-zona interaction. Although the specific sugar residue involved has not been characterized^[57], several candidates have been proposed, e.g., nonreducing -linked oligosaccharides^[58], -N-acetylglucosamine, mannose^[59,60] and fucose^[61,62].

Several proteins located on the sperm membrane implicated in gamete interaction have been reported. Galactosyltransferase(GTf) is a 60 Kd protein that uses the UDP galactose to link the sperm to the carbohydrate moiety of the ZP₃ glycoprotein^[63]. Another candidate to mediate the initial sperm-zona binding is the zonadhesin, a 16.4Kd protein synthesized exclusively in the testis and located at the apical region of the sperm head^[64]. Northern blot analysis shows that there is a restricted homology between zonadhesins from mice, pig and human. The pig zonadhesin interacts with the respective zona protein through two cystein residues found in the D0-D1 and D2-D4 domains. There are several molecules like sulfogalactosylglycerolipids^[65] or proteins that could interact with the egg envelope but most of them are not yet fully characterized^[66,67] and their function in fertilization is not known.

Both galactosyltransferase and sperm zonadhesin are implicated in acrosome-intact spermatozoa interaction with the zona pellucida's primary sperm receptor.

However, acrosome-reacted spermatozoa can also interact with another sperm receptor glycoprotein located in the zona pellucida. The secondary sperm receptor in the zona pellucida is the ZP₂ glycoprotein. It has been proposed that the protein moiety as well as the carbohydrates participate in the interaction. According to the model proposed by Hedrick and co-workers^[68] and supported by experimental evidences^[69,70], acrosin embedded in the inner acrosomal membrane interacts with the carbohydrates of ZP₂ by a lectin domain exposed by this acrosin type,whereas non-membrane bound acrosin actively digests the ZP₁ matrix. An equivalent model has been proposed for the guinea pig but in this case is a lectin-like site independent from the hyaluronidase site of the sperm surface PH-20 protein, that is involved in secondary sperm-egg interaction^[71]. An analog protein to PH20 has been found in bovine sperm^[13].

It has been reported that GnRH increases human sperm binding to homologous zona pellucida. This increase depends on extracellular Ca²⁺ transported through a L-type Ca²⁺ channel of the sperm plasma membrane^[72]. Once the sperm has traveled through the zona pellucida by a mechanical and protease-assisted activity, the acrosome-reacted sperm enters the perivitellyne space. Interaction between the equatorial segment of the sperm head and the egg plasma membrane compels the tangential orientation of the sperm and thus can interact with the egg plasmalema by the only plasma membrane covered region in the equatorial segment^[1].

Gamete membrane fusion has been associated to the interaction of several proteins. It has been proposed that gamete fusion is mediated by the interaction of anintegrin component in the egg plasma membrane with the heterodimeric fertilin in the sperm equatorial segment membrane^[73,74].  When fertilin  was disrupted in mice, fertility is highly affected^[75]. This is not the case with human sperm when fertilin gene is not functional^[76].

Several integrins types have been detected by immunofluorescence in guinea pig^[77], mouse^[73], pig^[78], and human eggs. When mouse eggs were pretreated with monoclonal or polyclonal antibodies against the 61 integrin and then exposed to sperm, less spermatozoa fuses^[73].

According to this model^[79,80], the binding domain of ADAM₂ in the sperm plasma membrane binds to the egg integrin through the disintegrin domain. This interaction induces a tridimensional change in the fusion domain (in the other subunit of ADAM₂) that permits their interaction with the hydrophobic surface of the egg plasmalemma. An hemifusion intermediate is formed when several fusogenic ADAM domains interact. This aggregation acts as a fusion pore allowing the entry of the sperm nucleus.

7  Concluding remarks

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Correspondence to: Dr. Hector Serrano, Dept. Health Sciences, UniversidadAutnoma Metropolitana-Iztapalapa, Ave. Michoacan and Purisima, Mexico City, DF 09340, Mexico.
Tel: +52-5-804 4733,     Fax: +52-5-804 4727
E-mail: hectorserrano@hotmail.com
Received 2001-10-19    Accepted 2001-11-21