The human acrosome reaction

H.W.G. Baker, D.Y. Liu, C. Garrett, M. Martic

University of Melbourne Department of Obstetrics and Gynaecology, Reproductive Services Melbourne IVF and Royal Women's Hospital, Carlton, Victoria 3053, Australia.

Asian J Androl  2000 Sep; 2: 172-178

Keywords: male infertility; fertilization in vitro; sperm-ovum interactions; zona pellucida;  acrosome reaction

Abstract

The observation that some patients with failure of fertilization had normal sperm-zona pellucida binding but no sperm-zona penetration led to the discovery of a specific defect of the zona pellucida induced acrosome reaction which causes severe chronic infertility^[3].

2 The normal fertilization process

The fertilising human sperm penetrates the cumulus oophorus and corona radiata with the acrosome intact, and binds to the surface of the zona pellucida by the plasma membrane overlying the acrosome. The binding reaction stimulates the acrosome reaction which occurs on the surface of the zona pellucida and continues during the early stages of sperm-zona pellucida penetration, but is complete by the time the sperm reaches the inner third of the zona pellucida. The sperm traverses the perivitelline space and binds to the oolemma. The initial contact is between the microvillous processes of the oocyte fusing with the plasma membrane of the equatorial segment of the sperm. This leads to engulfment of the sperm by the oocyte and triggers the cortical granule reaction which causes changes in the inner third of the zona pellucida and the oolemma, inhibiting further sperm gaining access to the ooplasm and thus preventing polyspermia^[4]. These events in the fertilization process are shown schematically in Figure 1.

Figure 1. Diagram of the events of human fertilization.

4 Role of the acrosome in sperm-zona pellucida binding

A method for examining sperm bound the surface of the zona pellucida was developed by crushing oocytes which had failed to fertilise between two glass slides after washing in protein free medium. This resulted in flattened zonae sticking to the glass slides, forming a sufficiently thin specimen to be stained by the Shorr method for examination of sperm morphology using standard light microscopy. The striking finding of this study was a higher proportion of sperm with normal morphology bound to the surface of the zona than that present in the insemination medium. The significantly lower proportions of various abnormal morphologies for the bound sperm indicated a powerful selection function of the zona pellucida for sperm with normal morphology^[11]. Some sperm with poor morphology did bind to the zona pellucida and it was interesting that the majority of these had normal acrosomal areas but were, for example, pyriform or small headed.

Study of the morphology of zona bound sperm has been extended by the development of a method to remove sperm from the surface of the zona pellucida (see below) for examination by light microscopy and computer assisted sperm morphometry^[12]. A number of morphometric parameters show dramatic selectivity by the sperm zona pellucida binding process. For example, the anterior area of the sperm head, identified by low optical density, reflects the area of the acrosome and a specific size (about 5 m²) is selected in sperm bound to the zona although there may be a wide range for the average sperm in the insemination medium. Other parameters associated with anterior symmetry of the sperm and width of the midpiece region at the junction with the head are also selected by zona binding^[13]. It is anticipated that further work to quantify the relationship between sperm morphometry and zona binding ability will improve the discrimination of fertile and subfertile men on the basis of sperm morphology. Knowing the characteristics of sperm with high zona binding ability may also improve the standard microscopic assessments of sperm morphology.

Although light microscopy and computer assisted morphometry can be used to assess the acrosomal region, the acrosome itself cannot be seen by standard light microscopic methods. However, labelled lectins or antibodies which bind to acrosomal components can be used to determine acrosome status, We have used either Pisum sativum agglutinin or a monoclonal antibody to a form of clusterin (E5) which is present in normal intact acrosomes^[1,14]. On a number of occasions we have found some relationship between the proportion of sperm with intact acrosomes in the insemination medium and the fertilization rates in vitro. Similarly, there is a relationship between the proportion of acrosome intact sperm in the medium and the numbers of sperm bound to the zona pellucida^[15-18]. These findings are consistent with the proposition that human sperm normally bind to the surface of the zona pellucida with the acrosome intact.

To test this proposition we artificially induced the acrosome reaction of human sperm with the calcium ionophore A23187. This agent is a powerful inducer of the acrosome reaction but also reduces sperm motility and therefore experiments had to be carefully performed to adjust the concentrations of acrosome intact and acrosome reacted motile sperm which were exposed to the oocytes. Using this technique we showed that the numbers of sperm bound to the oolemma of zona free oocytes increased as expected with increasing proportions of acrosome reacted sperm in the insemination medium, but the numbers of sperm bound to zona intact oocytes declined^[19]. This indicates that induction of the acrosome reaction in the insemination medium reduces or prevents sperm from binding to the zona pellucida. Although experimental models have been developed to show that acrosome reacting sperm are still capable of binding to the zona^[20], we believe the physiological fertilization process involves binding of acrosome intact sperm to the surface of the zona pellucida and the binding process stimulates the acrosome reaction to occur on the surface of the zona.

5 Role of the acrosome in sperm-zona pellucida penetration

There is also some controversy about the role of the acrosome reaction in sperm zona penetration. Some studies have shown that blocking the lytic action of acrosomal enzymes will prevent sperm zona binding but these may be criticised because the agents used may interfere with sperm motility. We conducted a study using soy bean trypsin inhibitor which reversibly blocks acrosin and were able to demonstrate a complete inhibition of sperm zona penetration and some interference with the acrosome reaction^[21]. We postulate that the enzyme inhibitor blocks dispersal of the acrosome matrix and that most of the acrosome matrix has to be removed before the sperm can penetrate the zona pellucida. Electron microscopy studies of oocytes indicate that sperm within the inner third of the zona are all completely acrosome reacted^[22].

6 Induction of the acrosome reaction by the zona pellucida

The acrosome can be lost from sperm in suspension. This spontaneous acrosome loss may be a feature of sperm senescence. Some inducers of the acrosome reaction, such as calcium ionophores, will augment acrosome loss in suspension but, as mentioned above, this is usually associated with a loss of sperm motility. In contrast, sperm which undergo the acrosome reaction on the surface of the zona, can often be removed from the zona and remain motile despite being acrosome reacted. Obviously the fertilising sperm, having bound and acrosome reacted on the zona, still requires motility to penetrate the zona, cross the perivitelline space and bind to the surface of the oolemma.

A number of studies of the acrosome reaction have been conducted using sperm in suspension and stimulators such as calcium ionophore, progesterone or prolonged incubation. There are reports of clinical conditions with low acrosome reaction rates and correlations between low spontaneous acrosome reaction or ionophore induced acrosome reaction and infertility or poor fertilization with standard IVF^[23,24]. We have also confirmed the latter^[25]. However, we have examined the relationship between ionophore and zona pellucida induced acrosome reactions with both intact and disaggregated zonae and found no such relation^[26]. Patients with poor zona induced acrosome reaction may have normal ionophore-induced acrosome reaction and vice versa^[26]. There was no relationship between ionophore-induced acrosome reaction and sperm zona pellucida penetration whereas zona pellucida-induced acrosome reaction and zona penetration were related^[27]. Thus it is important to use the physiological inducer, namely the zona pellucida, when studying the acrosome reaction. In the future, recombinant human ZP3 may become available for this purpose (Table 1).

7 Patients with normal sperm-zona pellucida binding but failure of sperm-zona pellucida penetration

As mentioned above, the majority of patients who have zero or low fertilization rates in vitro have poor sperm-zona pellucida binding and obvious defects of sperm quality such as poor sperm morphology. However, a subgroup of patients with poor fertilization were found to have a normal sperm-zona pellucida binding and further examination of these patients indicated that there was a failure of sperm-zona penetration^[3,7]. For these studies the oocytes were collected after they had failed to fertilise in vitro and sperm bound to the surface of the zona were removed by pipetting the oocytes through a fine bore glass tube with a slightly smaller internal diameter than that of the oocyte. The oocyte is deformed during the pipetting process and the surface bound sperm are sheared off. The sperm can be collected for further examination and the oocytes examined to count the number of sperm which have penetrated the zona pellucida or perivitelline space. Performing these procedures on patients with failure of fertilization but normal spermzona pellucida binding identified a specific defect of the acrosome reaction on the surface of the zona.

8 Discovery of disordered zona pellucida induced acrosome reaction

Patients with this condition generally have a long duration of primary infertility of unknown cause. In particular, the semen quality is generally good and the female partner has no major fertility problems (Table 3). With standard IVF the oocytes do not fertilise. Examination of these oocytes usually reveals many sperm bound to the surface of the zona, but the pipetting procedure reveals failure of sperm to penetrate the zona. When subsequently exposed to donor sperm the oocytes undergo normal sperm-zona pellucida binding, zona induced acrosome reaction and sperm penetration. In contrast, when sperm taken from these men are exposed to oocytes from other patients which have failed to fertilise in vitro the condition is reproduced. That is, the sperm bind normally but do not penetrate the zona pellucida. Removing the surface bound sperm and staining them with fluorescein labelled Pisum sativum agglutinin reveals that few have undergone the acrosome reaction. Our studies on the first 10 patients identified with this condition showed that the average number of zona bound sperm which underwent the acrosome reaction within two hours was about 6% compared with 60% in fertile controls^[3].

Table 3. Characteristics of disordered zona pellucida-induced acrosome reaction^[3].

Electron microscopy of oocytes from two patients who donated fresh oocytes for examination showed that four hours after insemination many sperm were bound to the surface of the zonae but they had all remained acrosome intact and the plasma membrane and outer acrosomal membrane appeared to be normal^[3]. This would suggest that the defect is situated in the signal transduction or effector pathways between the spermzona pellucida ligands and the fusion process between the plasma membrane and outer acrosomal membrane that starts the acrosome reaction^[4].

Since discovery of this condition in 1994 we have found over 50 patients with it. Some have been found by screening patients with idiopathic infertility. Others were diagnosed after standard IVF failed. We now recommend screening all patients with infertility of unknown cause and long duration for this condition before IVF is performed. If it is diagnosed, lCSl provides an effective treatment with high fertilization and pregnancy rates^[28,29].

9 Significance of the zona pellucida induced acrosome reaction

We have been studying the mechanism of the human Zona pellucida (ZP)-induced acrosome reaction in the hope of discovering the cause or causes of the defect. Although some parts of the signal transduction and effector pathways in the acrosome reaction have been discovered these may not be easy to demonstrate in our sperm-zona pellucida interaction tests^[30,31]. We have evidence that protein kinase C (PKC) is involved^[32]. There are either normal or impaired responses to stimulation of PKC with a phorbol ester in men with disordered zona pellucida-induced acrosome reaction suggesting and that there may be two groups of patients who have defects up and down stream of PKC. We have also found that inhibition of actin polymerization blocks the zona pellucida-induced acrosome reaction^[33]. We suspect that actin is involved in moving the plasma and outer acrosomal membranes together to facilitate fusion and initiate the release of the acrosomal contents. Whether some patients with disordered zona pellucida-induced acrosome reaction have abnormalities of this process remains to be discovered.

Acknowledgement 

This paper was presented in part in the Vishwa Nath Oration at the Second Asian and Oceanic Congress of Andrology, Chandigarh, India, 16-20 November, 1996.

References

[1] Liu DY, Baker HWG. Tests of human sperm function and fertilization in vitro. Fertil Steril 1992; 68: 465-83.
[2] Liu DY, Baker HWG. Defective sperm-zona pellucida interaction: a major cause of failure of fertilization in clinical in vitro fertilization.  Hum Reprod 2000; 15: 702-8.
[3] Liu DY, Baker HWG. Disordered acrosome reaction of sperm bound to the zona pellucida: a newly discovered sperm defect with reduced sperm-zona pellucida penetration and reduced fertilization in vitro. Hum Reprod 1994; 9: 1694-700.
[4] Yanagimachi R. Mammalian fertilization. In: Knobil E and Neill J, editors. The physiology of reproduction. 2nd edition. New York: Raven Press; 1994. p 189-317.
[5] Bourne H, Liu DY, Clarke GN, Baker HWG. Normal fertilization and embryo development by intracytoplasmic sperm injection of round-headed acrosomeless sperm. Fertil Steril 1995; 63: 1329-32.
[6] Nagy ZP, Liu J, Joris H, Verheyen G, Tournaye H, Camus M, et al. The result of intracytoplasmic sperm injection is not related to any of the three basic sperm parameters. Hum Reprod 1995; 10: 1123-9.
[7] Liu DY, Baker HWG. A new test for the assessment of sperm-zona pellucida penetration: relationship with results of other sperm tests and fertilization in vitro. Hum Reprod 1994; 9: 489-96.
[8] Van Blerkom J, Davis PW, Merriam J. A retrospective analysis of unfertilized and presumed parthenogentically activated human oocytes demonstrates a high frequency of sperm penetration. Hum Reprod, 1994; 9: 2381-8.
[9] Asch R, Simerly C, Ord T, Ord VA, Schatten G. The stages at which human fertilization arrests: microtubule and chromosome configuration in inseminated oocytes which failed to complete fertilization and development in humans. Hum Reprod 1995; 10: 1897-906.
[10] Liu DY, Clarke GN, Baker HWG. Inhibition of human sperm-zona pellucida and sperm-oolemma binding by antisperm antibodies. Fertil Steril 1991; 65: 440-2.
[11] Liu DY, Baker HWG. Morphology of spermatozoa bound to the zona pellucida of human oocytes that failed to fertilize in vitro. J Reprod Fert 1992; 94: 71-84.
[12] Garrett C, Baker HWG. A new fully automated system for the morphometric analysis of human sperm heads. Fertil Steril 1995; 63: 1306-17.
[13] Garrett C, Liu DY, Baker HWG. Selectivity of the human sperm-zona pellucida binding process to sperm head morphometry. Fertil Steril 1997; 67: 362-71.
[14] O'Byran MK, Murphy BF, Liu DY, Baker HWG. The use of anticlusterin monoclonal antibodies for the combined assessment of human sperm morphology and acrosome integrity. Hum Reprod 1994; 9: 1490-6.
[15] Liu DY, Baker HWG. The proportion of human sperm with poor morphology but normal intact acrosomes detected with pisum sativum agglutinin correlates with fertilization in vitro. Fertil Steril 1988; 50: 288-93.
[16] Liu DY, Baker HWG. Relationships between human sperm acrosin, acrosomes and fertilization in vitro. Hum Reprod 1990; 5: 298-303.
[17] Liu DY, Baker HWG. Acrosome status and morphology of human spermatozoa bound to the zona pellucida and oolemma determined using oocytes that failed to fertilize in vitro. Hum Reprod 1994; 9: 673-9.
[18] Liu DY, Lopata A, Johnston WIH, Baker HWG. Human sperm-zona pellucida binding, sperm characteristics and in vitro fertilization. Hum Reprod 1989; 4: 696-701.
[19] Liu DY, Baker HWG. Inducing the human acrosome reaction with a calcium ionophore A23187 decreases sperm-zona pellucida binding with oocytes that failed to fertilize in vitro. J Reprod Fert 1990; 89: 127-34.
[20] Morales P, Cross NL, Overstreet JW, Hanson FW. Acrosome-intact and acrosome-reacted human sperm can initiate binding to the zona pellucida. Dev Biol 1989; 133: 385-92.
[21] Liu DY, Baker HWG. Inhibition of acrosin activity with a trypsin inhibitor blocks human sperm penetration of the zona pellucida. Biol Reprod 1993; 48: 340-8.
[22] Sathananthan H, Ng S-C, Bongso A, Trounson A, Ratnam SS. Visual atlas of early human development for assisted reproductive technology. Singapore: National University of Singapore; 1993. p 83.
[23] Calvo L, Vantman D, Banks SM, Tezon J, Koukoulis GN, Dennison L, et al. Follicular fluid-induced acrosome reaction distinguishes a subgroup of men with unexplained infertility not identified by semen analysis. Fertil Steril 1989; 52: 1048-54.
[24] Cummins JM, Pember SM, Jequier AM, Yovich JL, Hartmann PE. A test of the human sperm acrosome reaction following ionophore challenge (ARIC). Relationship to fertility and other seminal parameters. J Androl 1991; 12: 98-103.
[25] Liu DY, Baker HWG. Calcium ionophore-induced acrosome reaction correlates with fertilization rates in vitro in patients with teratozoospermic semen. Hum Reprod 1998; 13: 905-10.
[26] Liu DY, Baker HWG. A simple method for assessment of the human acrosome reaction of spermatozoa bound to the zona pellucida: lack of relationship with ionophore A23187 induced acrosome reaction. Hum Reprod 1996; 11: 551-7.
[27] Liu DY, Baker HWG. Relationship between the zona pellucida (ZP) and ionophore A231 87-induced acrosome reaction and the ability of sperm to penetrate the ZP in men with normal sperm-ZP binding. Fertil Steril 1996; 66: 312-5.
[28] Liu DY, Bourne H, Baker HWG. Fertilization and pregnancy with acrosome intact sperm by intracytoplasmic sperm injection in patients with disordered zona pellucida induced acrosome reaction. Fertil Steril 1995; 64: 116-21.
[29] Liu DY, Bourne H, Baker HWG. High fertilization and pregnancy rates after intracytoplasmic sperm injection in patients with disordered zona pellucida-induced acrosome reaction. Fertil Steril 1997; 67: 955-8.
[30] Kopf GS, Gerton GL. The mammalian sperm acrosome and the acrosome reaction. In: Elements of mammalian fertilization. Wassarman PM, editor. Boston: CRC press; 1991. p 153-203.
[31] Breitbart H, Spungin B. The biochemistry of the acrosome reaction. Mol Hum Reprod 1997; 3: 195-202.
[32] Liu DY, Baker HWG.  Protein kinase C plays an important role in the human zona pellucida-induced acrosome reaction.  Mol Hum Reprod 1997; 3: 1037-43.
[33] Liu DY, Martic M, Clarke GN, Dunlop ME, Baker HWG. An important role of actin polymerization in the human zona pellucida-induced acrosome reaction.  Mol Hum Reprod 1999; 5: 941-9.
[34] World Health Organisation. WHO laboratory manual for examination of human semen and semen-cervical mucus interaction. Cambridge: Cambridge University Press; 1999.

Correspondence to: HWG Baker, MD, PhD, FRACP, Principal Research Fellow,University of Melbourne Department of Obstetrics and Gynaecology, The Royal Women's Hospital, Carlton 3053 Victoria, Australia.  
Work Phone: +61-3-9344 2130 or +61-3-9344 2620   
Fax: +61-3-9347 1761  Home Phone: +61-3-9819 5309  
e-mail: g.baker@obgyn-rwh.unimelb.edu.au
Received 2000-06-13     Accepted 2000-06-26