ANDROLOGIA
23, 205-208 ( 1991)
ACCEPTED: MARCH18, 1991
Electron microscopic evidence on the acrosomal status of bound sperm and their penetration into human hemizonae pellucida after storage in a buffered salt solution* D. R. Franken’, W. T . Oosthuizen’, S. Cooper2, T . F. Kruger’, L. J. Burkman3, C. C. Coddington3 and G. D. Hodgen3 Key words. Zona pellucida
~
sperm binding
~
sperm penetration
Summary. The hemizona assay (HZA) was developed to evaluate sperm binding potential using microbisected human zona pellucida. In this study, eight human oocytes stored in a buffered salt solution for 60 days were bisected into two identical hemispheres (hemizonae) and coincubated with the spermatozoa from a fertile man. All evaluated spermatozoa were tightly bound to the outer surface or had begun penetration into the zona pellucida. The hemizonae with bound spermatozoa were prepared and fixed for transmission electron microscopy (TEM) using standard techniques. Among the 108 sperm bound to the zone we were able to evaluate 25 by TEM. Twenty (80%) of the zona bound spermatozoa were partially or completely acrosome reacted, while six (20%) of the zona bound sperm had intact acrosomes. Acrosome intact, partially acrosome reacted and completely reacted spermatozoa were observed within the zona. Penetration
I Reproductive Biology Unit, Department of Obstetrics and Gynecology, University of Stellenbosch, Tygerberg Hospital, Typerberg, Department of Anatomical Pathology, Division of Electron Microscopy, Faculty of Medicine G71, University of the Orange Free State, Bloemfontein, South Africa, and ’ The Jones Insitute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, Virginia, USA.
Correspondence: Gary D. Hodgen, PhD, The Jones Institute for Reproductive Medicine, Eastern Virgina School of Medicine, 855 West Brambleton Ave., Suite B, Norfolk, Virginia 23510, USA.
*
This work was partially supported by the Contraceptive Research and Development Program (CONRAD), Eastern Virginia Medical School, under a Cooperative Agreement with the United States Agency for International Development (A.I.D.) DPE-2044-A-00-6063-00). The views expressed by the authors do not necessarily reflect the views of A.I.D.
~
electron microscopy
~
human.
pathways or tunnels were seen within the zona matrix. The results illustrate, that typically spermatozoa tightly bound the human zona pellucida show induction of the acrosome reaction. Importantly, following storage of human eggs in salt solution (buffered to 7.4), the zona pellucida retain their biological and functional characteristics for at least 90 days.
Introduction Sperm capacitation encompasses a critical series of functional changes which are accomplished either in vivo during transport through the female reproductive tract (Yanagimachi, 1988) or during in virro culture. Ideally, the clinical evaluation of fertility should document the ability of sperm to complete capacitation, the acrosome reaction and successfully penetrate the human zona pellucida. For that purpose, a number of “fertilization” assays are now in use, where the interaction of sperm and eggs can be assessed. These include the zona-free hamster ovum penetration assay (Yanagimachi et al., 1976), the intact zona penetration test (Overstreet et al., 1980), the hemizona sperm binding assay (Burkman et al., 1988) and, most recently, a test ofcompetitive sperm binding to a single intact zona using fluorochrome labels (Liu et al., 1989). The hemizona assay (HZA) is a homologous fertilization test which maximizes the use of scarce human zonae pellucida and controls for zona-to-zona variability. The two matching halves of a single zona pellucida are utilized for comparing the sperm binding capacities of a proven fertile specimen versus semen from a subfertile male. We have already demonstrated the assay feasibility and its usefulness in predicting fertilization failure (Burkman et al., 1988; Franken er a]., 1989a). More recently, we
206
D. R. FRANKEN ET AL.
have focused on certain physiological aspects of the binding assay and report here on electron microscopic observations of sperm interaction with the hemizona. In a separate manuscript, we have dealt with sperm hyperactivated motility and the freezing of sperm in relation to hemizona binding (Coddington et al., 1991). Our extensive use of a concentrated salt solution for zona storage in the HZA (Franken et al., 1989b) has been influenced by the previous reports (Yanagimachi et al., 1979; Yoshimatsu et al., 1988) concerning buffers in the storage fluid. We had separately observed that some zonae stored for 3-6 months in the unbuffered salt solution were quite fragile or had disintegrated (unpublished observations). In this paper, therefore, we have included data on pH alterations in the salt solutions with and without buffer during six months of storage.'
nesium chloride supplemented with 0.1 yo polyvinylpyrrolidone (PVP 40, Sigma St. Louis, MO). Solution B (buffered)(Yoshimatsu & Yanagimachi, 1988). Containing 1.5 mmol magnesium chloride supplemented with 0.1% PVP and 40 mmol Sodium hepes buffer (Boehringer, Germany). For both salt solution A and B, the pH was adjusted to 7.0 by the addition of a small quantity of 1 N NaOH. In Study 2, 0.5 ml aliquots of solution A and solution B were held in the oocyte storage tubes, but no oocytes were added. The vials were kept at + 4 "C or + 23 "C. For each treatment, the pH was noted on the initial day of storage, as well as 24 hours later. After 30 days, 120 days and 180 days of storage, three vials were opened per solution and the pH was again assessed. Sperm binding to the hemizona
Material and methods Oocyte collection and storage Immature human oocytes were obtained from surgically removed ovarian tissue which was thoroughly minced with blades (Burkman et al., 1988; Burkman et al., 1990). The pulp was layered onto a series of copper sieves (Scienceware, Bell-Art Products, Pequennock, NJ). The sieves were washed with 30 to 50 ml of phosphate buffered saline (PBS), allowing the fluid and oocytes to collect in plastic Petri dishes (Falcon Plastics =#3003, Becton Dickinson, Labware, Oxnard, CA). The viscous fluid containing the oocytes and cellular debris were transferred to conical tubes and centrifuged for 15 minutes at room temperature to allow the oocytes to settle to the bottom. Most of the supernatant PBS was discarded and the oocyte-bearing lower suspension was emptied into a clean Petri dish. Each dish was thoroughly scanned for oocytes using a dissecting microscope (Zeiss stereoscope DV, Germany). The collected oocytes were always placed in a small microfuge tube with 0.5 ml of a buffered salt solution (solution B) composed of 1.5 mmol magnesium chloride supplemented with 0.1 yo polyvinylpyrrolidone (PVP 40, Sigma, St. Louis, M O ) and 40 mmol sodium-hepes buffer (Boehringer, Germany) (Yanagimachi et al., 1979; Yoshimatsu et al., 1988). This salt solution containing the oocytes was stored at + 4 "C until used for sperm binding and T E M evaluation. Assessment o f p H changes during prolonged storage (Study 2) Solution A (unbuffered) (Yanagimachi et al., 1979; Franken et al., 1988). Composed of 1.5 mmol mag-
Handling of the sperm and oocytes for assessing tight binding to the hemizona followed the procedures previously published (Burkman et al., 1988). Briefly, twelve salt-stored oocytes were removed from the storage tubes, rinsed and carefully bisected into two hemispheres (hemizonae) using a micromanipulator (Narishige, Tokyo, Japan) and a fine microblade. Semen was obtained from one proven fertile man with normal semen characteristics (fathered a child during the previous two years). The sperm were washed twice with Ham's F10 medium +7.5yo human fetal cord serum, and allowed to swim out of the pellet during one hour of incubation at 37 "C. A portion of the supernatant sperm was diluted to a concentration of 500 000 motile ml-I. Each hemizona was placed in a separate 50 p1 droplet of the diluted sperm, the drop was covered with mineral oil, and coincubation was carried out for 4 hours (37 "C, 5% GO, in air). Each hemizona was then pipetted 5X to dislodge all loosely attached spermatozoa (Burkman et al., 1988). The number of spermatozoa that were firmly bound to the outer hemizona surface was counted under phase contrast microscopy (magnification x 200). The hemizonae were then placed directly into a PBS solution containing 3% glutaraldehyde for fixation. Electron microscopic preparation Micro-procedures were instituted which increased the efficiency of hemizona handling to minimize the possibility of loosing hemizonae. Following the primary fixation in glutaraldehyde, the hemizonae were washed in 0.2 mol PBS (pH 7.2) and postfixed for 90 minutes in a 1% Palade's buffered osmium tetroxide (Merck, Darmstadt, Germany). The hemizonae were dehydrated in a graded series of acetone ANDROLOGIA 23, 205-208 (1991)
ACROSOMAL STATUS OF
(20-100~0)and embedded singly in Spurr's epoxy resin (Merck, Darmstadt, Germany) using Beem capsules (Protea Laboratories, Johannesburg). During the acetone dehydration procedures, the acetone were changed instead of transferring the hemizonae at each step. Specially drawn micro pipettes were used to minimize hemizona loss. Sections of 60-80 nm were cut on a LKB ultramicrotome (Ultratone 111, Type 8802A, Beckman, Johannesburg) using a diamond knife (3 mm, angle 42 degrees, Diatome Ltd., Bienne, Switzerland). The sections were then stained with uranyl acetate and lead citrate. The microscopic examinations were carried out in a Phillips 30 1 (Phillips 3 1OG, Holland) electron microscope at 60 kV.
HEMIZONAL BOUND SPERM
207
Figure 1. Micrograph illustrating spermatozoa bound to hemizonae, magnification x 3 900. A and B: partially acrosome reacted; C: acrosome intact spermatozoon.
Results Electron microscopy The mean number ( f SD) of spermatozoa tightly bound to the hemizonae was 50.0 f 12. The observations on the number of bound sperm were recorded after the 4 hour coincubation and rinsing steps, prior to the onset of the T E M preparations. A total of 108 spermatozoa were found to be bound to or penetrating the hemizonae. Due to the limitations of sperm orientation and cutting angle, only 25 of the 108 spermatozoa could be evaluated for acrosomal status. Only those sperm where the acrosomal regions were clearly visible were included during the evaluation. Twenty (80%) clearly showed alterations of the acrosomal membranes: 14 (56%) were acrosome reacted, while 6 (24%) were partially acrosome reacted, showing a distinct ruffling of the acrosomal membranes (methods of Phillips, personal communications). Only 6 (24%) of the spermatozoa, were found intact acrosomes. Some acrosome-intact and partially intact spermatozoa had begun entry into the zona (Fig. 1 ) . Acrosome reacted spermatozoa were seen also in the zona, clearly leaving a penetration pathway or tunnel through the zona substance (Fig. 2). The recorded pH values (study 2) for the salt solution stored at different temperatures over a six month period are tabulated in Table 1. A sample of each solution was kept in a refrigerator for storage at 4 ° C and the remaining two samples were kept in a air conditioned room at 23°C. Storage temperature played no role in the pH stability of any of the two media tested as indicated in Table 1. Supplementation with 40 mmol hepes, stabilized the pH of solution B since the pH dropped only 0.2 units over the six month period. In contrast the unbuffered solution gradually became more acidic, with a drop of 2.2 units over the same period. ANDROLOGIA 23, 205-208 (1991)
Figure 2. Micrograph illustrating penetration pathways in zona matrix, magnification x 2 800. A and B: digestive pathway; C: inner margin of the zona pellucida.
Table 1. Storage temperatures play no role in the pH stability of the two media tested. Note the pH was stabilized when hepes buffer was added No. of MgCl,, PVP-40 days
+ Hepes
MgCl,, PVP-40
4 "C (pH)
23 "C (pH)
4 "C (pH) 23 "C (pH)
0 1 30 120 180
7.0 7.0 7.0 7.0 6.8
7.0 7.0 7.0 7.0 6.9
7.0 7.0 6.8 6.4 4.9
7.0 7.0 6.7 6.0 4.8
Mean
6.96 0.09
6.98 0.04
6.42 0.88
6.3 0.93
SD
Discussion The results of the present study and studies from others (Chen & Sathananthan, 1986; Cross et al., 1988; Coddington et al., 1990) illustrate the ability of the human zona pellucida to cause or in some cases enhance the induction of the acrosomal reaction. Intact sperm were firmly bound to the outer
208
D. R. FRANKENET AL.
surface of the zona by their plasma membranes. The plasma membrane and outer acrosomal membrane clearly showed a ruflled appearance just prior to vesiculation (Chen & Sathananathan, 1986). Acrosome intact, partially reacted and fully reacted spermatozoa had entered the zona 4 hours after insemination, but only fully reacted spermatozoa were seen in the inner zona area. We observed seven pathways created by spermatozoa as they penetrate through the zona pellucida. Only two of these pathways were associated with spermatozoa. These two spermatozoa were both within the inner zona and were fully acrosome reacted. The presence of the pathways confirms that the salt-stored zonae retain important functional aspects in that the spermatozoa can interact with the zona pellucida matrix, even after 3 months in the buffered salt solution (Yanagimachi et al., '1979; Franken et al., 1988; Yoshimatsu et al., 1988). Apart from retaining sperm binding properties, the zona glycoproteins will also permit spermatozoa to fully penetrate the zona thickness. During an earlier report, the sperm binding kinetics (Burkman et al., 1988) were characterized by a binding peak followed by a plateau or a decline in the number of bound spermatozoa. The decline in the sperm binding kinetic curve could have been caused, amont other reasons, by the fact that some of the bound spermatozoa were able to penetrate fully through the thickness of the zona pellucida. The pathways are likely the direct consequence of sperm penetration after the initial tight zona binding step has been completed (vigorous flagelIar movements or digestive action of the acrosomal enzymes). This phenomenon is clearly illustrated in Figure 2 and the observation is in close agreement with the early studies of Chen & Sathananthan (1986). The penetration pathways in the hemizonae might act as tunnels for following spermatozoa. This will eventually lead to a plateau in the sperm binding kinetics after a period of approximately 4 hours. We confirm and extend the findings of others that the major biological and biochemical properties of the zona pellucida are retained during salt storage (Yanagimachi et al., 1979). It has been advised to use neutralized salt solutions to prevent swelling and/or dissolving of the zona pellucida after prolonged storage (Yoshimatsu et al., 1988). Our data show good retention of sperm binding functional characteristics through 90 days of salt storage, wherein the majority of sperm bound to the zona pellucida matrix show partial or complete acrosome reaction in the HZA system.
References Burkman LJ, Coddington CC, Franken DR, Kruger TF, Rosenwaks Z, Hodgen G D (1988) The hemizona assay (HZA): Development of a diagnostic test for binding of human spermatozoa to human hemizona pellucida to predict fertilization potential. Fertil Steril 49:688-697 Burkman LJ, Coddington CC, Franken DR, Oehninger SC, Hodgen G D (1990) The Hemizona assay (HZA): Assessment of fertilizing potential by means of human sperm binding to the human zona pellucida. In: Laboratory Diagnosis and Treatment of Infertility. Keel BA, Welester BW (eds) CRC Press, Boca Raton, pp 2 13-228 Chen C, Sathananathan AH (1986) Early penetration of human sperm through the vestments of human eggs in vitro. Arch Androl 16:183-1 97 Coddington CC, Fulgham DL, Alexander NJ, Johnson D, Herr J, Hodgen G D (1990) Sperm bound to the zona pellucida in hemizona assay (HZA) demonstrate acrosome reaction (AR) when stained with T-6 antibody. Fertil Steril 54:504-508 Coddington CC, Franken DR, Burkman LJ, Oosthuizen WT, Kruger T, Hodgen G D (1991) Functional aspects of human sperm binding to the zona pellucida using the hemizona assay. J Androl 12:l-8 Cross NL, Morales P, Overstreet JW, Hanson FW (1988) Induction of the acrosome reaction by the human zona pellucida. Biol Reprod 38:235-244 Franken DR, Burkman LJ, Oehninger SC, Coddington CC, Veeck L, Kruger TF, Rosenwaks Z, Hodgen G D (1989a) The hemizona assay: using salt stored human oocytes: Evaluation of zona pellucida capacity for binding spermatozoa. Gamete Res 22: 15-26 Franken DR, Oehninger SC, Burkman LJ, Coddington CC, Kruger TF, Rosenwaks Z,Acosta AA, Hodgen GD (1989b) The Hemizona Assay (HZA): A predictor of human sperm fertilizing potential in in virro fertilization treatment. J In Vitro Fert Embryo Transfer 6:44-50 Liu DY, Clarke GN, Lopata A, Johnston WIH, Baker HWG (1989) A sperm-zona pellucida binding test and in vitro fertilization. Fertil Steril 52281-287 Overstreet JW, Yanagimachi R, Katz DF, Hayashi K, Hanson FW (1980) Penetration ofhuman spermatozoa into the human zona pellucida and the zona-free hamster egg: a study of fertile donors and infertile patients. Fertil Steril 33:534-542 Yanagimachi R , Yanagimachi H, Rogers J (1976) The use of zona-free animal ova as a test system for the assessment of the fertilizing capacity of human spermatozoa. Biol Reprod 15: 47 1-476 Yanagimachi R , Lopata A, Odom CD, Bronson RA, Mahi CA, Micholson GL (1979) Retention of biologic characteristics of zona pellucida in highly concentrated salt solutions: The use of salt storage eggs for assessing the fertilizing capacity of spermatozoa. Fertil Steril 31:562-574 Yanagimachi R ( 1988) Mammalian fertilization. In: Physiology of Reproduction. Knobil E, Neil1 J (eds) Raven Press, New York, pp 135-186 Yoshimatsu R, Yanagimachi R, Zopta A (1988) Zonae pellucidae of salt stored hamster and human eggs: their penetratibility by homologous and heterologous spermatozoa. Gamete Res 21:115-126
ANDROLOGIA 23, 205-208 (1991)
23, 205-208 ( 1991)
ACCEPTED: MARCH18, 1991
Electron microscopic evidence on the acrosomal status of bound sperm and their penetration into human hemizonae pellucida after storage in a buffered salt solution* D. R. Franken’, W. T . Oosthuizen’, S. Cooper2, T . F. Kruger’, L. J. Burkman3, C. C. Coddington3 and G. D. Hodgen3 Key words. Zona pellucida
~
sperm binding
~
sperm penetration
Summary. The hemizona assay (HZA) was developed to evaluate sperm binding potential using microbisected human zona pellucida. In this study, eight human oocytes stored in a buffered salt solution for 60 days were bisected into two identical hemispheres (hemizonae) and coincubated with the spermatozoa from a fertile man. All evaluated spermatozoa were tightly bound to the outer surface or had begun penetration into the zona pellucida. The hemizonae with bound spermatozoa were prepared and fixed for transmission electron microscopy (TEM) using standard techniques. Among the 108 sperm bound to the zone we were able to evaluate 25 by TEM. Twenty (80%) of the zona bound spermatozoa were partially or completely acrosome reacted, while six (20%) of the zona bound sperm had intact acrosomes. Acrosome intact, partially acrosome reacted and completely reacted spermatozoa were observed within the zona. Penetration
I Reproductive Biology Unit, Department of Obstetrics and Gynecology, University of Stellenbosch, Tygerberg Hospital, Typerberg, Department of Anatomical Pathology, Division of Electron Microscopy, Faculty of Medicine G71, University of the Orange Free State, Bloemfontein, South Africa, and ’ The Jones Insitute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, Virginia, USA.
Correspondence: Gary D. Hodgen, PhD, The Jones Institute for Reproductive Medicine, Eastern Virgina School of Medicine, 855 West Brambleton Ave., Suite B, Norfolk, Virginia 23510, USA.
*
This work was partially supported by the Contraceptive Research and Development Program (CONRAD), Eastern Virginia Medical School, under a Cooperative Agreement with the United States Agency for International Development (A.I.D.) DPE-2044-A-00-6063-00). The views expressed by the authors do not necessarily reflect the views of A.I.D.
~
electron microscopy
~
human.
pathways or tunnels were seen within the zona matrix. The results illustrate, that typically spermatozoa tightly bound the human zona pellucida show induction of the acrosome reaction. Importantly, following storage of human eggs in salt solution (buffered to 7.4), the zona pellucida retain their biological and functional characteristics for at least 90 days.
Introduction Sperm capacitation encompasses a critical series of functional changes which are accomplished either in vivo during transport through the female reproductive tract (Yanagimachi, 1988) or during in virro culture. Ideally, the clinical evaluation of fertility should document the ability of sperm to complete capacitation, the acrosome reaction and successfully penetrate the human zona pellucida. For that purpose, a number of “fertilization” assays are now in use, where the interaction of sperm and eggs can be assessed. These include the zona-free hamster ovum penetration assay (Yanagimachi et al., 1976), the intact zona penetration test (Overstreet et al., 1980), the hemizona sperm binding assay (Burkman et al., 1988) and, most recently, a test ofcompetitive sperm binding to a single intact zona using fluorochrome labels (Liu et al., 1989). The hemizona assay (HZA) is a homologous fertilization test which maximizes the use of scarce human zonae pellucida and controls for zona-to-zona variability. The two matching halves of a single zona pellucida are utilized for comparing the sperm binding capacities of a proven fertile specimen versus semen from a subfertile male. We have already demonstrated the assay feasibility and its usefulness in predicting fertilization failure (Burkman et al., 1988; Franken er a]., 1989a). More recently, we
206
D. R. FRANKEN ET AL.
have focused on certain physiological aspects of the binding assay and report here on electron microscopic observations of sperm interaction with the hemizona. In a separate manuscript, we have dealt with sperm hyperactivated motility and the freezing of sperm in relation to hemizona binding (Coddington et al., 1991). Our extensive use of a concentrated salt solution for zona storage in the HZA (Franken et al., 1989b) has been influenced by the previous reports (Yanagimachi et al., 1979; Yoshimatsu et al., 1988) concerning buffers in the storage fluid. We had separately observed that some zonae stored for 3-6 months in the unbuffered salt solution were quite fragile or had disintegrated (unpublished observations). In this paper, therefore, we have included data on pH alterations in the salt solutions with and without buffer during six months of storage.'
nesium chloride supplemented with 0.1 yo polyvinylpyrrolidone (PVP 40, Sigma St. Louis, MO). Solution B (buffered)(Yoshimatsu & Yanagimachi, 1988). Containing 1.5 mmol magnesium chloride supplemented with 0.1% PVP and 40 mmol Sodium hepes buffer (Boehringer, Germany). For both salt solution A and B, the pH was adjusted to 7.0 by the addition of a small quantity of 1 N NaOH. In Study 2, 0.5 ml aliquots of solution A and solution B were held in the oocyte storage tubes, but no oocytes were added. The vials were kept at + 4 "C or + 23 "C. For each treatment, the pH was noted on the initial day of storage, as well as 24 hours later. After 30 days, 120 days and 180 days of storage, three vials were opened per solution and the pH was again assessed. Sperm binding to the hemizona
Material and methods Oocyte collection and storage Immature human oocytes were obtained from surgically removed ovarian tissue which was thoroughly minced with blades (Burkman et al., 1988; Burkman et al., 1990). The pulp was layered onto a series of copper sieves (Scienceware, Bell-Art Products, Pequennock, NJ). The sieves were washed with 30 to 50 ml of phosphate buffered saline (PBS), allowing the fluid and oocytes to collect in plastic Petri dishes (Falcon Plastics =#3003, Becton Dickinson, Labware, Oxnard, CA). The viscous fluid containing the oocytes and cellular debris were transferred to conical tubes and centrifuged for 15 minutes at room temperature to allow the oocytes to settle to the bottom. Most of the supernatant PBS was discarded and the oocyte-bearing lower suspension was emptied into a clean Petri dish. Each dish was thoroughly scanned for oocytes using a dissecting microscope (Zeiss stereoscope DV, Germany). The collected oocytes were always placed in a small microfuge tube with 0.5 ml of a buffered salt solution (solution B) composed of 1.5 mmol magnesium chloride supplemented with 0.1 yo polyvinylpyrrolidone (PVP 40, Sigma, St. Louis, M O ) and 40 mmol sodium-hepes buffer (Boehringer, Germany) (Yanagimachi et al., 1979; Yoshimatsu et al., 1988). This salt solution containing the oocytes was stored at + 4 "C until used for sperm binding and T E M evaluation. Assessment o f p H changes during prolonged storage (Study 2) Solution A (unbuffered) (Yanagimachi et al., 1979; Franken et al., 1988). Composed of 1.5 mmol mag-
Handling of the sperm and oocytes for assessing tight binding to the hemizona followed the procedures previously published (Burkman et al., 1988). Briefly, twelve salt-stored oocytes were removed from the storage tubes, rinsed and carefully bisected into two hemispheres (hemizonae) using a micromanipulator (Narishige, Tokyo, Japan) and a fine microblade. Semen was obtained from one proven fertile man with normal semen characteristics (fathered a child during the previous two years). The sperm were washed twice with Ham's F10 medium +7.5yo human fetal cord serum, and allowed to swim out of the pellet during one hour of incubation at 37 "C. A portion of the supernatant sperm was diluted to a concentration of 500 000 motile ml-I. Each hemizona was placed in a separate 50 p1 droplet of the diluted sperm, the drop was covered with mineral oil, and coincubation was carried out for 4 hours (37 "C, 5% GO, in air). Each hemizona was then pipetted 5X to dislodge all loosely attached spermatozoa (Burkman et al., 1988). The number of spermatozoa that were firmly bound to the outer hemizona surface was counted under phase contrast microscopy (magnification x 200). The hemizonae were then placed directly into a PBS solution containing 3% glutaraldehyde for fixation. Electron microscopic preparation Micro-procedures were instituted which increased the efficiency of hemizona handling to minimize the possibility of loosing hemizonae. Following the primary fixation in glutaraldehyde, the hemizonae were washed in 0.2 mol PBS (pH 7.2) and postfixed for 90 minutes in a 1% Palade's buffered osmium tetroxide (Merck, Darmstadt, Germany). The hemizonae were dehydrated in a graded series of acetone ANDROLOGIA 23, 205-208 (1991)
ACROSOMAL STATUS OF
(20-100~0)and embedded singly in Spurr's epoxy resin (Merck, Darmstadt, Germany) using Beem capsules (Protea Laboratories, Johannesburg). During the acetone dehydration procedures, the acetone were changed instead of transferring the hemizonae at each step. Specially drawn micro pipettes were used to minimize hemizona loss. Sections of 60-80 nm were cut on a LKB ultramicrotome (Ultratone 111, Type 8802A, Beckman, Johannesburg) using a diamond knife (3 mm, angle 42 degrees, Diatome Ltd., Bienne, Switzerland). The sections were then stained with uranyl acetate and lead citrate. The microscopic examinations were carried out in a Phillips 30 1 (Phillips 3 1OG, Holland) electron microscope at 60 kV.
HEMIZONAL BOUND SPERM
207
Figure 1. Micrograph illustrating spermatozoa bound to hemizonae, magnification x 3 900. A and B: partially acrosome reacted; C: acrosome intact spermatozoon.
Results Electron microscopy The mean number ( f SD) of spermatozoa tightly bound to the hemizonae was 50.0 f 12. The observations on the number of bound sperm were recorded after the 4 hour coincubation and rinsing steps, prior to the onset of the T E M preparations. A total of 108 spermatozoa were found to be bound to or penetrating the hemizonae. Due to the limitations of sperm orientation and cutting angle, only 25 of the 108 spermatozoa could be evaluated for acrosomal status. Only those sperm where the acrosomal regions were clearly visible were included during the evaluation. Twenty (80%) clearly showed alterations of the acrosomal membranes: 14 (56%) were acrosome reacted, while 6 (24%) were partially acrosome reacted, showing a distinct ruffling of the acrosomal membranes (methods of Phillips, personal communications). Only 6 (24%) of the spermatozoa, were found intact acrosomes. Some acrosome-intact and partially intact spermatozoa had begun entry into the zona (Fig. 1 ) . Acrosome reacted spermatozoa were seen also in the zona, clearly leaving a penetration pathway or tunnel through the zona substance (Fig. 2). The recorded pH values (study 2) for the salt solution stored at different temperatures over a six month period are tabulated in Table 1. A sample of each solution was kept in a refrigerator for storage at 4 ° C and the remaining two samples were kept in a air conditioned room at 23°C. Storage temperature played no role in the pH stability of any of the two media tested as indicated in Table 1. Supplementation with 40 mmol hepes, stabilized the pH of solution B since the pH dropped only 0.2 units over the six month period. In contrast the unbuffered solution gradually became more acidic, with a drop of 2.2 units over the same period. ANDROLOGIA 23, 205-208 (1991)
Figure 2. Micrograph illustrating penetration pathways in zona matrix, magnification x 2 800. A and B: digestive pathway; C: inner margin of the zona pellucida.
Table 1. Storage temperatures play no role in the pH stability of the two media tested. Note the pH was stabilized when hepes buffer was added No. of MgCl,, PVP-40 days
+ Hepes
MgCl,, PVP-40
4 "C (pH)
23 "C (pH)
4 "C (pH) 23 "C (pH)
0 1 30 120 180
7.0 7.0 7.0 7.0 6.8
7.0 7.0 7.0 7.0 6.9
7.0 7.0 6.8 6.4 4.9
7.0 7.0 6.7 6.0 4.8
Mean
6.96 0.09
6.98 0.04
6.42 0.88
6.3 0.93
SD
Discussion The results of the present study and studies from others (Chen & Sathananthan, 1986; Cross et al., 1988; Coddington et al., 1990) illustrate the ability of the human zona pellucida to cause or in some cases enhance the induction of the acrosomal reaction. Intact sperm were firmly bound to the outer
208
D. R. FRANKENET AL.
surface of the zona by their plasma membranes. The plasma membrane and outer acrosomal membrane clearly showed a ruflled appearance just prior to vesiculation (Chen & Sathananathan, 1986). Acrosome intact, partially reacted and fully reacted spermatozoa had entered the zona 4 hours after insemination, but only fully reacted spermatozoa were seen in the inner zona area. We observed seven pathways created by spermatozoa as they penetrate through the zona pellucida. Only two of these pathways were associated with spermatozoa. These two spermatozoa were both within the inner zona and were fully acrosome reacted. The presence of the pathways confirms that the salt-stored zonae retain important functional aspects in that the spermatozoa can interact with the zona pellucida matrix, even after 3 months in the buffered salt solution (Yanagimachi et al., '1979; Franken et al., 1988; Yoshimatsu et al., 1988). Apart from retaining sperm binding properties, the zona glycoproteins will also permit spermatozoa to fully penetrate the zona thickness. During an earlier report, the sperm binding kinetics (Burkman et al., 1988) were characterized by a binding peak followed by a plateau or a decline in the number of bound spermatozoa. The decline in the sperm binding kinetic curve could have been caused, amont other reasons, by the fact that some of the bound spermatozoa were able to penetrate fully through the thickness of the zona pellucida. The pathways are likely the direct consequence of sperm penetration after the initial tight zona binding step has been completed (vigorous flagelIar movements or digestive action of the acrosomal enzymes). This phenomenon is clearly illustrated in Figure 2 and the observation is in close agreement with the early studies of Chen & Sathananthan (1986). The penetration pathways in the hemizonae might act as tunnels for following spermatozoa. This will eventually lead to a plateau in the sperm binding kinetics after a period of approximately 4 hours. We confirm and extend the findings of others that the major biological and biochemical properties of the zona pellucida are retained during salt storage (Yanagimachi et al., 1979). It has been advised to use neutralized salt solutions to prevent swelling and/or dissolving of the zona pellucida after prolonged storage (Yoshimatsu et al., 1988). Our data show good retention of sperm binding functional characteristics through 90 days of salt storage, wherein the majority of sperm bound to the zona pellucida matrix show partial or complete acrosome reaction in the HZA system.
References Burkman LJ, Coddington CC, Franken DR, Kruger TF, Rosenwaks Z, Hodgen G D (1988) The hemizona assay (HZA): Development of a diagnostic test for binding of human spermatozoa to human hemizona pellucida to predict fertilization potential. Fertil Steril 49:688-697 Burkman LJ, Coddington CC, Franken DR, Oehninger SC, Hodgen G D (1990) The Hemizona assay (HZA): Assessment of fertilizing potential by means of human sperm binding to the human zona pellucida. In: Laboratory Diagnosis and Treatment of Infertility. Keel BA, Welester BW (eds) CRC Press, Boca Raton, pp 2 13-228 Chen C, Sathananathan AH (1986) Early penetration of human sperm through the vestments of human eggs in vitro. Arch Androl 16:183-1 97 Coddington CC, Fulgham DL, Alexander NJ, Johnson D, Herr J, Hodgen G D (1990) Sperm bound to the zona pellucida in hemizona assay (HZA) demonstrate acrosome reaction (AR) when stained with T-6 antibody. Fertil Steril 54:504-508 Coddington CC, Franken DR, Burkman LJ, Oosthuizen WT, Kruger T, Hodgen G D (1991) Functional aspects of human sperm binding to the zona pellucida using the hemizona assay. J Androl 12:l-8 Cross NL, Morales P, Overstreet JW, Hanson FW (1988) Induction of the acrosome reaction by the human zona pellucida. Biol Reprod 38:235-244 Franken DR, Burkman LJ, Oehninger SC, Coddington CC, Veeck L, Kruger TF, Rosenwaks Z, Hodgen G D (1989a) The hemizona assay: using salt stored human oocytes: Evaluation of zona pellucida capacity for binding spermatozoa. Gamete Res 22: 15-26 Franken DR, Oehninger SC, Burkman LJ, Coddington CC, Kruger TF, Rosenwaks Z,Acosta AA, Hodgen GD (1989b) The Hemizona Assay (HZA): A predictor of human sperm fertilizing potential in in virro fertilization treatment. J In Vitro Fert Embryo Transfer 6:44-50 Liu DY, Clarke GN, Lopata A, Johnston WIH, Baker HWG (1989) A sperm-zona pellucida binding test and in vitro fertilization. Fertil Steril 52281-287 Overstreet JW, Yanagimachi R, Katz DF, Hayashi K, Hanson FW (1980) Penetration ofhuman spermatozoa into the human zona pellucida and the zona-free hamster egg: a study of fertile donors and infertile patients. Fertil Steril 33:534-542 Yanagimachi R , Yanagimachi H, Rogers J (1976) The use of zona-free animal ova as a test system for the assessment of the fertilizing capacity of human spermatozoa. Biol Reprod 15: 47 1-476 Yanagimachi R , Lopata A, Odom CD, Bronson RA, Mahi CA, Micholson GL (1979) Retention of biologic characteristics of zona pellucida in highly concentrated salt solutions: The use of salt storage eggs for assessing the fertilizing capacity of spermatozoa. Fertil Steril 31:562-574 Yanagimachi R ( 1988) Mammalian fertilization. In: Physiology of Reproduction. Knobil E, Neil1 J (eds) Raven Press, New York, pp 135-186 Yoshimatsu R, Yanagimachi R, Zopta A (1988) Zonae pellucidae of salt stored hamster and human eggs: their penetratibility by homologous and heterologous spermatozoa. Gamete Res 21:115-126
ANDROLOGIA 23, 205-208 (1991)
