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Localization of fibronectin on the surface of human spermatozoa and relation to the sperm-egg interaction
Vol. 61, No.3, March 1994
FERTILITY AND STERILITY
Printed on acid-free paper in U. S. A.
Copyright" 1994 The American Fertility Society
Localization of fibronectin on the surface of human spermatozoa and relation to the sperm-egg interaction
Kazuhiko Hoshi, M.D., Ph.D.*t Hiroko Sasaki, M.D., Ph.D.* Kaoru Yanagida, M.D., Ph.D.*
Akira Sato, M.D., Ph.D.* Akira Tsuiki, M.D., Ph.D.:!:
Fukushima Medical College, Fukushima, and National MitoHospital, Mito, Japan
Objectives: The mechanism of human sperm-oolemma adhesion and penetration as well as localization of fibronectin on the sperm head and its relation to fertilization were investigated. Design: Sperm-oolemma interaction was examined with an in vitro assay of the human spermzona-free hamster egg interaction. Localization of fibronectin on the surface of human spermatozoa was observed by the back scattered electron imaging mode of the scanning electron microscopy (SEM). Results: It was confirmed by observations under SEM that the anterior tip of the sperm head is the first to come into contact with the egg plasma membrane but that the equatorial segment of the sperm head is the first to be trapped by microvilli of the plasma membrane and that the postacrosomal region is first incorporated into ooplasm. Localization of fibronectin on the equatorial segment of the human sperm head was detected by SEM. Antifibronectin antibodies inhibited human sperm-oolemma adhesion significantly. Conclusions: Important involvement of fibronectin in the gamete interaction was made clear by the fact that fibronectin is localized in the region where a spermatozoon is fused first with the egg plasma membrane during fertilization and that the sperm adhering to the egg is inhibited by antifibronectin antibodies. Fertil Steril 1994;61:542-7 Key Words: Spermatozoa, fertilization, fibronectin, oocytes
Fertilization requires maturation of gametes and capacitation, acrosome reaction, and hyperactivation of the spermatozoa. A spermatozoon satisfying the conditions reaches the oocyte surface, passes through the zona pellucida (ZP), and becomes attached to the egg plasma membrane. In the mammal, sperm -egg fusion begins between the sperm head plasma membrane and the egg plasma mem-
Received June 8, 1993; revised and accepted November 10, 1993. * Department of Obstetrics and Gynecology, Fukushima Medical College. t Reprint requests: Kazuhiko Hoshi, M.D., DepartmentofObstetrics and Gyne.cology, Fukushima Medical College, 1-Hikarigaoka, Fukushima 960-12, Japan. Department of Obstetrics and Gynecology, National Mito Hospital.
*
542
Hoshi et aI.
Fibronectin on human spermatozoa
brane. After sperm-egg fusion, sperm and egg pronuclei are formed, and both pronuclei are fused to complete fertilization. The region of the mammal sperm that is fused first with the egg plasma membrane is said to be the equatorial segment of the sperm head (1), and it has been suggested that a special mechanism to accelerate the sperm -oolemma adhesion is present in this region. Primarily, evaluation of the fertilizing ability of human spermatozoa should be studied by the fertilization system using human eggs. However, Yanagimachi et aL (2) have reported that evaluation can be made by in vitro assay of the human sperm-zona-free hamster egg interaction. Using this system, we observed the process of human spermatozoa being adhered to and penetrated into the plasma membrane by scanning electron microscopy (SEM) and observed localization of fibronectin, Fertility and Sterility
one of the cell-adhering factors on the surface of spermatozoa by the back scattered electron imaging mode of the SEM to investigate the role of fibronectin in fertilization.
MATERIALS AND METHODS Human Sperm-Zona-Free Hamster Egg Interaction Observed by SEM Preparation of the Sperm Suspension
Semen samples were obtained from fertile donors by masturbation and allowed to stand at room temperature until liquefaction was completed. A swimup method was used to collect vigorously motile spermatozoa from the semen samples. The liquefied semen was divided into 0.5-mL aliquots and then transferred to the bottom of the test tubes containing 2.0 mL of modified Biggers, Whitten, and Whittingham (BWW) medium (3) supplemented with 0.3% human serum albumin (Fraction V powder; Sigma Chemical Co., St. Louis, MO). The tubes were inclined to an angle of 30° and incubated in an atmosphere of 5% CO 2 in air at 37°C for 1 hour. During this time, vigorously motile spermatozoa swam upward out of the seminal plasma and into the modified BWW medium. This suspension was centrifuged for 5 minutes at 500 X g. The pellet was resuspended in a very small amount of modified BWW medium and diluted to obtain adequate concentration for insemination. Collection of the Zona-Free Hamster Eggs
Eggs were obtained from superovulated female golden hamsters (90 to 150 g). To induce superovulation, 30 IV of pregnant mare serum gonadotropin (Sigma Chemical Co.) was given intraperitoneally, and then 30 IV of hCG (HCG Mochida; Mochida, Tokyo, Japan). Cumulus-oocyte masses were dissected from the oviducts 17 hours after hCG administration, and the cumulus cells were dispersed in 0.1 % hyaluronidase. After washing two times with modified BWW medium, the eggs were placed in 0.1 % trypsin to remove the ZP. Finally, zona-free eggs were washed three times with fresh modified BWW medium and kept at 37°C in air before use. Gamete Interaction In Vitro
Sperm suspension was placed in a plastic Petri dish containing mineral oil. After the sperm were preincubated, the zona-free hamster eggs were put Vol. 61, No.3, March 1994
into the sperm suspension. The final sperm concentration was from 5 to 8 X 106 jmL. The preparation was kept at 37°C under 5% CO 2 in air. At various times after the insemination, eggs were examined by electron microscope. Preparation of Oocyte for SEM
Samples were transferred to a fixative consisting of 0.2% phosphate buffer solution (pH 7.4) containing 2.5% glutaraldehyde in a refrigerator (4°C) for 12 hours and then washed with 0.2% phosphate buffer and distilled water. Eggs were placed in a special glass container and quickly dehydrated in graded ethanol series (50%, 60%, 70%, 80%, 90%, 95%, and 99%) and then put into isoamyl acetate. Critical point drying was performed with a Hitachi HCP-1 critical point apparatus (Hitachi, Tokyo, Japan). After mounting on the aluminum slab, metal coating was performed with gold in a vaccuum evaporator. Observation was made with a Hitachi S-700 scanning electron microscope (Hitachi). Observations on Localization of Fibronectin on the Surface of Human Spermatozoa Preparation of the Sperm Suspension
Semen samples were obtained from fertile donors. A swim-up method was used to collect suitable spermatozoa. Vigorously motile spermatozoa were transferred to 1.0 mL of modified BWW medium and incubated in 5% CO 2 in air at 37°C for 6 hours. This suspension was centrifuged at 300 X g for 5 minutes, washed with 0.2% phosphate-buffered solution (PBS) (pH 7.2), and adjusted so that the final sperm concentration was 5 X 107 jmL. This preparation was used as a sample for SEM. Preparation of Spermatozoa for SEM
Several drops of the sperm suspension were mounted on slides coated with polylysin, and the slides were immersed in a 4°C Zamboni's solution (4) for 20 minutes for fixation. After washing with 80% ethanol, they were rinsed three times with Dulbecco's PBS and then put into normal goat serum at 4°C for blocking. The smears were incubated with 0.1 % anti-human fibronectin rabbit antibodies (Wako, Tokyo, Japan) for 24 hours at room temperature. After washing in PBS, the slides were incubated with secondary antibodies labeled with the colloidal gold (Auro-Probe EM GARIgG; Janssen Life Sciences Products, Beerse, Belgium) for 30 Hoshi et a1.
Fibronectin on human spermatozoa
543
Figure 1 Scanning electron micrographs of human sperm-zona free hamster egg interaction. Anterior tip of sperm head was trapped by elongated microvilli (A). Microvilli were observed adjacent to the plasma membrane over the equatorial segment (B). Equatorial segment was covered with cytoplasmic proorusions of the ovum (C). Bar, 1 I'm.
minutes at 37°C. After dehydrating in graded ethanol series, the samples were subjected to the critical point drying and carbon coated for observations by the back scattered electron imaging mode of the SEM using a Hitachi S-800 scanning electron microscope (Hitachi).
concentration was 2 X 106 /mL. The preparation was kept at 37°C under 5% CO 2 in air. After incubation for 2 hours, the eggs were mounted on a slide with a coverslide and observed under a phase-contrast microscope to determine sperm adhesion to the surface of the eggs.
Effect of Antifibronectin Antibody on the Human Sperm-Zona-Free Hamster Egg Interaction
Human Sperm Penetration Test into Zona-Free Hamster Egg
Preparation of the Sperm Suspension
Semen samples were obtained from fertile donors. A swim-up method was used to collect suitable spermatozoa. Vigorously motile spermatozoa were transferred to 1.0 mL of modified BWW medium and incubated in 5% CO 2 in air at 37°C for 5 hours. After that, spermatozoa were incubated with modified BWW medium containing 20% (vol/vol) anti-human fibronectin rabbit antibodies for another 2 hours (preincubation). Controls consisted of spermatozoa incubated with modified BWW medium containing 20% normal rabbit serum (Cedarlane Laboratories, Ltd., Hornby, Ontario, Canada).
After preincubation, sperm suspension was placed in a Petri dish containing mineral oil. Zonafree hamster eggs were put into the sperm suspension. The final sperm concentration was 2 X 107 / mL. After incubation at 37°C under 5% CO 2 in air for 6 hours, the eggs were mounted on a slide with a coverslide and observed under a phase-contrast microscope to determine sperm penetration. The egg was evaluated as "penetrated" when either a swollen sperm head or a male pronucleus with sperm tail was found in the cytoplasm. Student's t-test and x2 test were used to analyze for statistical differences between groups.
RESULTS
Human Sperm Adherence Test to Zona-Free Hamster Egg
After the spermatozoa were preincubated, sperm suspension was placed in a plastic Petri dish containing mineral oil. Zona-free hamster eggs were put into the sperm suspension. The final sperm 544
Hoshi et at.
Fibronectin on human spermatozoa
Human Sperm-Zona-Free Hamster Egg Interaction
The surface of the oocyte is covered with numerous microvilli that are evenly and densely distribFertility and Sterility
Figure 2 Scanning electron micrographs of human sperm observed by the back scattered electron imaging mode . Localization of colloidal gold was observed on the equatorial segment of the acrosomeintact sperm head. (A), The back scattered electron imaging mode. (B), The secondary electron imaging mode. Localization of colloidal gold was observed on the equatorial segment of the acrosomereacted sperm head. (C), The back scattered electron imaging mode. (D), The secondary electron imaging mode. Bar, 1 /L m .
uted on the vitellus surface. Most of the spermatozoa attached to the oocyte surface have completed the acrosome reaction. After the initial contact with the surface of the ovum, the sperm head was trapped by elongated microvilli. Initially, the microvilli appeared to grasp and immobilize the anterior tip of the sperm (Fig. IA). As gamete interaction proceeded, the microVol. 61, No.3, March 1994
villi appeared to trap the sperm head at the region of the equatorial segment (Fig. IB). In the later stage, there were microvillar portions on the plasma membrane of equatorial segment. The equatorial segment was covered with cytoplasmic protrusions of the ovum (Fig. Ie). Such spermatozoa may be partially submerged into the ooplasm. The postacrosomal region seemed to be first incorporated Hoshi et al.
Fibronectin on human spermatozoa
545
Table 1 Effect of Antifibronectin Antibody on Adhesion of Zona-Free Hamster Eggs by Human Spermatozoa
Antifibronectin antibody
No. of experiments
No. of eggs inseminated
No. of oolemmaladherent sperm*
3 3
31 43
4.56 ± 2.62t 7.44 ± 3.61
+ - (control)
* Values are means ± SD. t p < 0.01 compared with control.
into the ooplasm. The anterior tip of the sperm head was the last portion to be incorporated. Observation of Fibronectin on the Surface of Human Spermatozoa
Acrosome-reacted and unreacted spermatozoa were observed. Colloidal gold was clearly visualized as a minute granule that was 15 nm in diameter and that had very high electron density. Localization of colloidal gold was found on the equatorial segment of the sperm head regardless of whether or not the acrosome reaction occurred (Fig. 2). The localization was hardly observed in the regions other than the equatorial segment. As a control, spermatozoa were exposed to a secondary antibody in the absence of primary antibodies, but colloidal gold was not observed on the sperm surface. Effect of Antifibronectin Antibody on the Human Sperm-Zona-Free Hamster Egg Interaction
Human Sperm Adherence Test to Zona-Free Hamster Egg
The number of spermatozoa adhered to zona-free hamster eggs was 4.56 ± 2.62 (mean ± SD) in the presence of antifibronectin antibodies. This was a significant decrease (P < 0.01) compared with 7.44 ± 3.61 of the control (Table 1). Human Sperm Penetration Test into Zona-Free Hamster Egg
The penetration rate of human spermatozoa into zona-free hamster eggs was 34% in the presence of antifibronectin antibodies, which was not significantly different from 35% of control. The mean number of penetrating spermatozoa into a zonafree egg was 0.53 in the presence of antifibronectin antibodies, which was not significantly different from 0.53 of control (Table 2). 546
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DISCUSSION
Upon undergoing the acrosome reaction and hyperactivation, the spermatozoa passed through the ZP to reach the oocyte surface. The region where the acrosome-reacted spermatozoa is fused first with the egg plasma membrane is said to be the equatorial segment of the sperm head (1). In the present study that observed the process of human spermatozoa penetrating the zona-free hamster egg by SEM, it was shown that the contact with the oocyte surface starts from the anterior tip of the sperm head. The equatorial segment of the sperm is the first to be trapped by microvilli on the oocyte surface, suggesting that the sperm-egg fusion proceeds in humans according to the same mechanism as seen in other mammals. Fertilization consists essentially of cell-to-cell fusion of a spermatozoon with an ovum. It is believed that adhesion molecules are concerned with the process of the cell fusion (5). Many substances are known as the adhesion molecules. Glander et al. (6) and Vuento et al. (7) have reported that fibronectin, one of the adhesion molecules, is present in the sperm head. Fibronectin is a high -molecular weight (MW) glycoprotein polypeptide (MW 250,000) consisting of two identical chains connected by disulfied bridges, and it can exist in two formsplasma fibronectin and cellular fibronectin. The two are similar in the physiological function, having the cell adhesion activity and versatile binding affinities for various biological substances. They are known to combine with approximately 20 kinds of high -MW proteins such as collagen, fibrin, heparin' DNA, ganglioside, microbes, and component of complement C1q. Fibronectin is formed by six different domains; its binding affinity for cells is detected in only domain IV, and its binding affinity site is the ARGG L Y -ASP amino acid sequence (tripeptide RG D) (8-10). It is also known that the fibronectin receptor of plasma membrane recognizes RGD. Table 2 Effect of Antifibronectin Antibody on Penetration of Zona-Free Hamster Eggs by Human Spermatozoa
Antifibronectin antibody
No. of experiments
Eggs penetrated
5 5
34 (36/107)* 35 (36/104)
Penetrating sperm per egg
%
+ - (control)
0.53 0.53
* Values in parentheses are the number of eggs penetrated per the total number of eggs. Fertility and Sterility
Observations on fibronectin in the spermatozoa have hitherto been made with a light microscope (11,12). We examined the localization of fibronectin by the back scattered electron imaging mode of the SEM. Fibronectin was found localized on the E)quatorial segment of sperm surface. The equatorial segment is a part of the sperm that penetrates into the egg first. The localization of fibronectin in this region suggests that fibronectin is concerned with the sperm-egg interaction. The effect of antifibronectin antibody on the sperm-egg interaction was examined by in vitro assay of the human sperm-zona-free hamster egg interaction. The antibody had no effect on the sperm penetration rate but inhibited the sperm-oolemmal adhesion significantly. These results indicate that fibronectin is concerned not with the sperm-egg fusion but with the sperm -egg adhesion. Immunoglobulin G-Fc receptor, a substance other than fibronectin that is concerned with the sperm-egg interaction, has been confirmed recently in the plasma membrane of hamster and human (13). The presence of C3 receptor has been reported in spermatozoa of some mammals (Anderson DJ, Wang H, Jack RM, abstract). Also, a report says that ARG-GLY-ASP (RGD)-containing oligopeptide, the cell binding affinity site of fibronectin, inhibits the human sperm-hamster egg adhesion and penetration. The presence of the RGD receptor on the surface of spermatozoa has also been suggested (14). There is also a report that C1q, a component of the classical complement pathway accelerates the human sperm-hamster egg adhesion but inhibits fusion (15). C1q is one of the high-MW proteins that combine with fibronectin (16), so this substance along with RGD attracts much attention in relation to fibronectin. REFERENCES 1. Yanagimachi R. Mammalian fertilization. In: Knobil E, Neil DJ, editors. The physiology of reproduction. New York: Raven Press Ltd., 1988:135-85.
Vol. 61, No.3, March 1994
2. Yanagimachi R, Yanagimachi H, Roger BJ. The use of zona-free animal ova as a test system for the fertilizing capacity of human spermatozoa. BioI Reprod 1976;15:471-6. 3. Overstreet JW, Yanagimachi R, Katz DF, Hayashi K, Hanson FW. Penetration of human spermatozoa into the human rona pellucida and the zona-free hamster egg. A study of fertile donors and infertile patients. Fertil Steril 1980;33:534-42. 4. Stefanini M, DeMartio C, Zamboni C. Fixation of ejaculated spermatozoa for electron microscopy. Nature 1967; 216:173-4. 5. Fuang TTF Jr, Yanagimachi R. Inner acrosomal membrane of mammalian spermatozoa: its properties and possible functions in fertilization. In: Koehler JK, editor. Gamete surfaces and their interactions. New York: Alan R Liss Inc., 1985:249-68. 6. Glander HJ, Herrmann K, Haustein UFo The equatorial fibronectin band (FEB) on human spermatozoa-a diagnostic help for male fertility? Andrologia 1987;19:456-9. 7. Vuento M, KuuselaP, Virkki M, Koskimies A. Characterization of fibronectin on human spermatozoa. Hoppe-Seyler's Z Physiol Chern 1984;365:757-62. 8. Hayashi M, Yamada KM. Domain structure of the carboxyl-terminal half of human plasma fibronectin. J BioI Chern 1983;258:3332-40. 9. Sekiguchi K, Hakomori S. Functional domain structure of fibronectin. Proc Natl Acad Sci 1980;77:2661-5. 10. Pierschbacher MD, Ruoslahti E. Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule. Nature 1984;309:30-3. 11. Harven E, Soligo D. Scanning electron microscopy of the surface antigens labeled with colloidal gold. Am J Anat 1986;175:277"':87. 12. Fusi FM, Bronson RA. Sperm surface fibronectin:' expression following capacitation. J Androl 1992;13:28-35. 13. Bronson RA, Fleit HB, Fusi F. Identification of an oolemmal IgG Fc receptor: its role in promoting binding of antibody-labelled human sperm to zona-free hamster eggs. Am J Reprod ImmunoI1990;23:87-92. 14. Bronson RA, Fusi F. Sperm-oolemmal interaction: role of the ARG-GLY-ASP (RGD) adhesion peptide. Fertil Steril 1990;54:527-9. 15. Fusi F, Bronson RA, Hong Y, Ghebrehiwet B. Complement component C1q and its receptor are involved in the interaction of human sperm with zona-free hamster eggs. Mol Reprod Dev 1991;29:180-8. 16. Reid KBM. Proteins involved in the activation and control of the two pathways of human complement. Biochem Soc Trans 1983;11:1-12.
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Fibronectin on human spermatozoa
547
FERTILITY AND STERILITY
Printed on acid-free paper in U. S. A.
Copyright" 1994 The American Fertility Society
Localization of fibronectin on the surface of human spermatozoa and relation to the sperm-egg interaction
Kazuhiko Hoshi, M.D., Ph.D.*t Hiroko Sasaki, M.D., Ph.D.* Kaoru Yanagida, M.D., Ph.D.*
Akira Sato, M.D., Ph.D.* Akira Tsuiki, M.D., Ph.D.:!:
Fukushima Medical College, Fukushima, and National MitoHospital, Mito, Japan
Objectives: The mechanism of human sperm-oolemma adhesion and penetration as well as localization of fibronectin on the sperm head and its relation to fertilization were investigated. Design: Sperm-oolemma interaction was examined with an in vitro assay of the human spermzona-free hamster egg interaction. Localization of fibronectin on the surface of human spermatozoa was observed by the back scattered electron imaging mode of the scanning electron microscopy (SEM). Results: It was confirmed by observations under SEM that the anterior tip of the sperm head is the first to come into contact with the egg plasma membrane but that the equatorial segment of the sperm head is the first to be trapped by microvilli of the plasma membrane and that the postacrosomal region is first incorporated into ooplasm. Localization of fibronectin on the equatorial segment of the human sperm head was detected by SEM. Antifibronectin antibodies inhibited human sperm-oolemma adhesion significantly. Conclusions: Important involvement of fibronectin in the gamete interaction was made clear by the fact that fibronectin is localized in the region where a spermatozoon is fused first with the egg plasma membrane during fertilization and that the sperm adhering to the egg is inhibited by antifibronectin antibodies. Fertil Steril 1994;61:542-7 Key Words: Spermatozoa, fertilization, fibronectin, oocytes
Fertilization requires maturation of gametes and capacitation, acrosome reaction, and hyperactivation of the spermatozoa. A spermatozoon satisfying the conditions reaches the oocyte surface, passes through the zona pellucida (ZP), and becomes attached to the egg plasma membrane. In the mammal, sperm -egg fusion begins between the sperm head plasma membrane and the egg plasma mem-
Received June 8, 1993; revised and accepted November 10, 1993. * Department of Obstetrics and Gynecology, Fukushima Medical College. t Reprint requests: Kazuhiko Hoshi, M.D., DepartmentofObstetrics and Gyne.cology, Fukushima Medical College, 1-Hikarigaoka, Fukushima 960-12, Japan. Department of Obstetrics and Gynecology, National Mito Hospital.
*
542
Hoshi et aI.
Fibronectin on human spermatozoa
brane. After sperm-egg fusion, sperm and egg pronuclei are formed, and both pronuclei are fused to complete fertilization. The region of the mammal sperm that is fused first with the egg plasma membrane is said to be the equatorial segment of the sperm head (1), and it has been suggested that a special mechanism to accelerate the sperm -oolemma adhesion is present in this region. Primarily, evaluation of the fertilizing ability of human spermatozoa should be studied by the fertilization system using human eggs. However, Yanagimachi et aL (2) have reported that evaluation can be made by in vitro assay of the human sperm-zona-free hamster egg interaction. Using this system, we observed the process of human spermatozoa being adhered to and penetrated into the plasma membrane by scanning electron microscopy (SEM) and observed localization of fibronectin, Fertility and Sterility
one of the cell-adhering factors on the surface of spermatozoa by the back scattered electron imaging mode of the SEM to investigate the role of fibronectin in fertilization.
MATERIALS AND METHODS Human Sperm-Zona-Free Hamster Egg Interaction Observed by SEM Preparation of the Sperm Suspension
Semen samples were obtained from fertile donors by masturbation and allowed to stand at room temperature until liquefaction was completed. A swimup method was used to collect vigorously motile spermatozoa from the semen samples. The liquefied semen was divided into 0.5-mL aliquots and then transferred to the bottom of the test tubes containing 2.0 mL of modified Biggers, Whitten, and Whittingham (BWW) medium (3) supplemented with 0.3% human serum albumin (Fraction V powder; Sigma Chemical Co., St. Louis, MO). The tubes were inclined to an angle of 30° and incubated in an atmosphere of 5% CO 2 in air at 37°C for 1 hour. During this time, vigorously motile spermatozoa swam upward out of the seminal plasma and into the modified BWW medium. This suspension was centrifuged for 5 minutes at 500 X g. The pellet was resuspended in a very small amount of modified BWW medium and diluted to obtain adequate concentration for insemination. Collection of the Zona-Free Hamster Eggs
Eggs were obtained from superovulated female golden hamsters (90 to 150 g). To induce superovulation, 30 IV of pregnant mare serum gonadotropin (Sigma Chemical Co.) was given intraperitoneally, and then 30 IV of hCG (HCG Mochida; Mochida, Tokyo, Japan). Cumulus-oocyte masses were dissected from the oviducts 17 hours after hCG administration, and the cumulus cells were dispersed in 0.1 % hyaluronidase. After washing two times with modified BWW medium, the eggs were placed in 0.1 % trypsin to remove the ZP. Finally, zona-free eggs were washed three times with fresh modified BWW medium and kept at 37°C in air before use. Gamete Interaction In Vitro
Sperm suspension was placed in a plastic Petri dish containing mineral oil. After the sperm were preincubated, the zona-free hamster eggs were put Vol. 61, No.3, March 1994
into the sperm suspension. The final sperm concentration was from 5 to 8 X 106 jmL. The preparation was kept at 37°C under 5% CO 2 in air. At various times after the insemination, eggs were examined by electron microscope. Preparation of Oocyte for SEM
Samples were transferred to a fixative consisting of 0.2% phosphate buffer solution (pH 7.4) containing 2.5% glutaraldehyde in a refrigerator (4°C) for 12 hours and then washed with 0.2% phosphate buffer and distilled water. Eggs were placed in a special glass container and quickly dehydrated in graded ethanol series (50%, 60%, 70%, 80%, 90%, 95%, and 99%) and then put into isoamyl acetate. Critical point drying was performed with a Hitachi HCP-1 critical point apparatus (Hitachi, Tokyo, Japan). After mounting on the aluminum slab, metal coating was performed with gold in a vaccuum evaporator. Observation was made with a Hitachi S-700 scanning electron microscope (Hitachi). Observations on Localization of Fibronectin on the Surface of Human Spermatozoa Preparation of the Sperm Suspension
Semen samples were obtained from fertile donors. A swim-up method was used to collect suitable spermatozoa. Vigorously motile spermatozoa were transferred to 1.0 mL of modified BWW medium and incubated in 5% CO 2 in air at 37°C for 6 hours. This suspension was centrifuged at 300 X g for 5 minutes, washed with 0.2% phosphate-buffered solution (PBS) (pH 7.2), and adjusted so that the final sperm concentration was 5 X 107 jmL. This preparation was used as a sample for SEM. Preparation of Spermatozoa for SEM
Several drops of the sperm suspension were mounted on slides coated with polylysin, and the slides were immersed in a 4°C Zamboni's solution (4) for 20 minutes for fixation. After washing with 80% ethanol, they were rinsed three times with Dulbecco's PBS and then put into normal goat serum at 4°C for blocking. The smears were incubated with 0.1 % anti-human fibronectin rabbit antibodies (Wako, Tokyo, Japan) for 24 hours at room temperature. After washing in PBS, the slides were incubated with secondary antibodies labeled with the colloidal gold (Auro-Probe EM GARIgG; Janssen Life Sciences Products, Beerse, Belgium) for 30 Hoshi et a1.
Fibronectin on human spermatozoa
543
Figure 1 Scanning electron micrographs of human sperm-zona free hamster egg interaction. Anterior tip of sperm head was trapped by elongated microvilli (A). Microvilli were observed adjacent to the plasma membrane over the equatorial segment (B). Equatorial segment was covered with cytoplasmic proorusions of the ovum (C). Bar, 1 I'm.
minutes at 37°C. After dehydrating in graded ethanol series, the samples were subjected to the critical point drying and carbon coated for observations by the back scattered electron imaging mode of the SEM using a Hitachi S-800 scanning electron microscope (Hitachi).
concentration was 2 X 106 /mL. The preparation was kept at 37°C under 5% CO 2 in air. After incubation for 2 hours, the eggs were mounted on a slide with a coverslide and observed under a phase-contrast microscope to determine sperm adhesion to the surface of the eggs.
Effect of Antifibronectin Antibody on the Human Sperm-Zona-Free Hamster Egg Interaction
Human Sperm Penetration Test into Zona-Free Hamster Egg
Preparation of the Sperm Suspension
Semen samples were obtained from fertile donors. A swim-up method was used to collect suitable spermatozoa. Vigorously motile spermatozoa were transferred to 1.0 mL of modified BWW medium and incubated in 5% CO 2 in air at 37°C for 5 hours. After that, spermatozoa were incubated with modified BWW medium containing 20% (vol/vol) anti-human fibronectin rabbit antibodies for another 2 hours (preincubation). Controls consisted of spermatozoa incubated with modified BWW medium containing 20% normal rabbit serum (Cedarlane Laboratories, Ltd., Hornby, Ontario, Canada).
After preincubation, sperm suspension was placed in a Petri dish containing mineral oil. Zonafree hamster eggs were put into the sperm suspension. The final sperm concentration was 2 X 107 / mL. After incubation at 37°C under 5% CO 2 in air for 6 hours, the eggs were mounted on a slide with a coverslide and observed under a phase-contrast microscope to determine sperm penetration. The egg was evaluated as "penetrated" when either a swollen sperm head or a male pronucleus with sperm tail was found in the cytoplasm. Student's t-test and x2 test were used to analyze for statistical differences between groups.
RESULTS
Human Sperm Adherence Test to Zona-Free Hamster Egg
After the spermatozoa were preincubated, sperm suspension was placed in a plastic Petri dish containing mineral oil. Zona-free hamster eggs were put into the sperm suspension. The final sperm 544
Hoshi et at.
Fibronectin on human spermatozoa
Human Sperm-Zona-Free Hamster Egg Interaction
The surface of the oocyte is covered with numerous microvilli that are evenly and densely distribFertility and Sterility
Figure 2 Scanning electron micrographs of human sperm observed by the back scattered electron imaging mode . Localization of colloidal gold was observed on the equatorial segment of the acrosomeintact sperm head. (A), The back scattered electron imaging mode. (B), The secondary electron imaging mode. Localization of colloidal gold was observed on the equatorial segment of the acrosomereacted sperm head. (C), The back scattered electron imaging mode. (D), The secondary electron imaging mode. Bar, 1 /L m .
uted on the vitellus surface. Most of the spermatozoa attached to the oocyte surface have completed the acrosome reaction. After the initial contact with the surface of the ovum, the sperm head was trapped by elongated microvilli. Initially, the microvilli appeared to grasp and immobilize the anterior tip of the sperm (Fig. IA). As gamete interaction proceeded, the microVol. 61, No.3, March 1994
villi appeared to trap the sperm head at the region of the equatorial segment (Fig. IB). In the later stage, there were microvillar portions on the plasma membrane of equatorial segment. The equatorial segment was covered with cytoplasmic protrusions of the ovum (Fig. Ie). Such spermatozoa may be partially submerged into the ooplasm. The postacrosomal region seemed to be first incorporated Hoshi et al.
Fibronectin on human spermatozoa
545
Table 1 Effect of Antifibronectin Antibody on Adhesion of Zona-Free Hamster Eggs by Human Spermatozoa
Antifibronectin antibody
No. of experiments
No. of eggs inseminated
No. of oolemmaladherent sperm*
3 3
31 43
4.56 ± 2.62t 7.44 ± 3.61
+ - (control)
* Values are means ± SD. t p < 0.01 compared with control.
into the ooplasm. The anterior tip of the sperm head was the last portion to be incorporated. Observation of Fibronectin on the Surface of Human Spermatozoa
Acrosome-reacted and unreacted spermatozoa were observed. Colloidal gold was clearly visualized as a minute granule that was 15 nm in diameter and that had very high electron density. Localization of colloidal gold was found on the equatorial segment of the sperm head regardless of whether or not the acrosome reaction occurred (Fig. 2). The localization was hardly observed in the regions other than the equatorial segment. As a control, spermatozoa were exposed to a secondary antibody in the absence of primary antibodies, but colloidal gold was not observed on the sperm surface. Effect of Antifibronectin Antibody on the Human Sperm-Zona-Free Hamster Egg Interaction
Human Sperm Adherence Test to Zona-Free Hamster Egg
The number of spermatozoa adhered to zona-free hamster eggs was 4.56 ± 2.62 (mean ± SD) in the presence of antifibronectin antibodies. This was a significant decrease (P < 0.01) compared with 7.44 ± 3.61 of the control (Table 1). Human Sperm Penetration Test into Zona-Free Hamster Egg
The penetration rate of human spermatozoa into zona-free hamster eggs was 34% in the presence of antifibronectin antibodies, which was not significantly different from 35% of control. The mean number of penetrating spermatozoa into a zonafree egg was 0.53 in the presence of antifibronectin antibodies, which was not significantly different from 0.53 of control (Table 2). 546
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Fibronectin on human spermatozoa
DISCUSSION
Upon undergoing the acrosome reaction and hyperactivation, the spermatozoa passed through the ZP to reach the oocyte surface. The region where the acrosome-reacted spermatozoa is fused first with the egg plasma membrane is said to be the equatorial segment of the sperm head (1). In the present study that observed the process of human spermatozoa penetrating the zona-free hamster egg by SEM, it was shown that the contact with the oocyte surface starts from the anterior tip of the sperm head. The equatorial segment of the sperm is the first to be trapped by microvilli on the oocyte surface, suggesting that the sperm-egg fusion proceeds in humans according to the same mechanism as seen in other mammals. Fertilization consists essentially of cell-to-cell fusion of a spermatozoon with an ovum. It is believed that adhesion molecules are concerned with the process of the cell fusion (5). Many substances are known as the adhesion molecules. Glander et al. (6) and Vuento et al. (7) have reported that fibronectin, one of the adhesion molecules, is present in the sperm head. Fibronectin is a high -molecular weight (MW) glycoprotein polypeptide (MW 250,000) consisting of two identical chains connected by disulfied bridges, and it can exist in two formsplasma fibronectin and cellular fibronectin. The two are similar in the physiological function, having the cell adhesion activity and versatile binding affinities for various biological substances. They are known to combine with approximately 20 kinds of high -MW proteins such as collagen, fibrin, heparin' DNA, ganglioside, microbes, and component of complement C1q. Fibronectin is formed by six different domains; its binding affinity for cells is detected in only domain IV, and its binding affinity site is the ARGG L Y -ASP amino acid sequence (tripeptide RG D) (8-10). It is also known that the fibronectin receptor of plasma membrane recognizes RGD. Table 2 Effect of Antifibronectin Antibody on Penetration of Zona-Free Hamster Eggs by Human Spermatozoa
Antifibronectin antibody
No. of experiments
Eggs penetrated
5 5
34 (36/107)* 35 (36/104)
Penetrating sperm per egg
%
+ - (control)
0.53 0.53
* Values in parentheses are the number of eggs penetrated per the total number of eggs. Fertility and Sterility
Observations on fibronectin in the spermatozoa have hitherto been made with a light microscope (11,12). We examined the localization of fibronectin by the back scattered electron imaging mode of the SEM. Fibronectin was found localized on the E)quatorial segment of sperm surface. The equatorial segment is a part of the sperm that penetrates into the egg first. The localization of fibronectin in this region suggests that fibronectin is concerned with the sperm-egg interaction. The effect of antifibronectin antibody on the sperm-egg interaction was examined by in vitro assay of the human sperm-zona-free hamster egg interaction. The antibody had no effect on the sperm penetration rate but inhibited the sperm-oolemmal adhesion significantly. These results indicate that fibronectin is concerned not with the sperm-egg fusion but with the sperm -egg adhesion. Immunoglobulin G-Fc receptor, a substance other than fibronectin that is concerned with the sperm-egg interaction, has been confirmed recently in the plasma membrane of hamster and human (13). The presence of C3 receptor has been reported in spermatozoa of some mammals (Anderson DJ, Wang H, Jack RM, abstract). Also, a report says that ARG-GLY-ASP (RGD)-containing oligopeptide, the cell binding affinity site of fibronectin, inhibits the human sperm-hamster egg adhesion and penetration. The presence of the RGD receptor on the surface of spermatozoa has also been suggested (14). There is also a report that C1q, a component of the classical complement pathway accelerates the human sperm-hamster egg adhesion but inhibits fusion (15). C1q is one of the high-MW proteins that combine with fibronectin (16), so this substance along with RGD attracts much attention in relation to fibronectin. REFERENCES 1. Yanagimachi R. Mammalian fertilization. In: Knobil E, Neil DJ, editors. The physiology of reproduction. New York: Raven Press Ltd., 1988:135-85.
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2. Yanagimachi R, Yanagimachi H, Roger BJ. The use of zona-free animal ova as a test system for the fertilizing capacity of human spermatozoa. BioI Reprod 1976;15:471-6. 3. Overstreet JW, Yanagimachi R, Katz DF, Hayashi K, Hanson FW. Penetration of human spermatozoa into the human rona pellucida and the zona-free hamster egg. A study of fertile donors and infertile patients. Fertil Steril 1980;33:534-42. 4. Stefanini M, DeMartio C, Zamboni C. Fixation of ejaculated spermatozoa for electron microscopy. Nature 1967; 216:173-4. 5. Fuang TTF Jr, Yanagimachi R. Inner acrosomal membrane of mammalian spermatozoa: its properties and possible functions in fertilization. In: Koehler JK, editor. Gamete surfaces and their interactions. New York: Alan R Liss Inc., 1985:249-68. 6. Glander HJ, Herrmann K, Haustein UFo The equatorial fibronectin band (FEB) on human spermatozoa-a diagnostic help for male fertility? Andrologia 1987;19:456-9. 7. Vuento M, KuuselaP, Virkki M, Koskimies A. Characterization of fibronectin on human spermatozoa. Hoppe-Seyler's Z Physiol Chern 1984;365:757-62. 8. Hayashi M, Yamada KM. Domain structure of the carboxyl-terminal half of human plasma fibronectin. J BioI Chern 1983;258:3332-40. 9. Sekiguchi K, Hakomori S. Functional domain structure of fibronectin. Proc Natl Acad Sci 1980;77:2661-5. 10. Pierschbacher MD, Ruoslahti E. Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule. Nature 1984;309:30-3. 11. Harven E, Soligo D. Scanning electron microscopy of the surface antigens labeled with colloidal gold. Am J Anat 1986;175:277"':87. 12. Fusi FM, Bronson RA. Sperm surface fibronectin:' expression following capacitation. J Androl 1992;13:28-35. 13. Bronson RA, Fleit HB, Fusi F. Identification of an oolemmal IgG Fc receptor: its role in promoting binding of antibody-labelled human sperm to zona-free hamster eggs. Am J Reprod ImmunoI1990;23:87-92. 14. Bronson RA, Fusi F. Sperm-oolemmal interaction: role of the ARG-GLY-ASP (RGD) adhesion peptide. Fertil Steril 1990;54:527-9. 15. Fusi F, Bronson RA, Hong Y, Ghebrehiwet B. Complement component C1q and its receptor are involved in the interaction of human sperm with zona-free hamster eggs. Mol Reprod Dev 1991;29:180-8. 16. Reid KBM. Proteins involved in the activation and control of the two pathways of human complement. Biochem Soc Trans 1983;11:1-12.
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