Human Reproduction vol.7 no 7 pp.999-1003, 1992
Is the human oocyte plasma membrane polarized?
Luigia Santella1, Mina Alikani2, Beth E.Talansky2, Jacques Cohen2-3 and Brian Dale1 'Stazione Zoologica, Villa Communale I, 80121, Napoli, Italy and The Gamete and Embryo Research Laboratory, The Center for Reproductive Medicine and Infertility, Department of Obstetrics and Gynecology, The New York Hospital—Cornell University Medical College, PO Box 30, 1300 York Avenue, New York, NY 10021, USA
membrane is organized in regularly spaced, short microvilli, except for an area overlying the metaphase spindle, which is essentially flat (Johnson etal., 1975; Shalgi and Phillips,
2
3
To whom corrspondence should be addressed
Using scanning electron microscopy, we have shown that the plasma membrane of the human metaphase II oocyte is organized in evenly spaced, short microvilli of 1 - 3 fim in length. In contrast to other mammals studied to date, there is no microvillus-free area overlying the metaphase spindle and there were no other indications of polarization at this level of organization. Functional polarity of the plasma membrane, studied using localized microsurgery of the zona pelhicida followed by insemination, suggests that sperm fusion and entry in the human may occur anywhere over the oocyte surface. Aged oocytes and those exposed to acidic Tyrode's solution had surfaces which were not homogeneously covered by microvilli. Oocytes exposed to a sucrose solution and subzonally injected with spermatozoa showed evidence of partial cortical granule exocytosis. Key words: first polar body/regional micromanipulation/ microvillus-free areayactivationypartial cortical exocytosis
Introduction In many animals, sperm entry is localized to a specific region of the egg surface (Monroy, 1965). This regionalization may be found at the level of the extracellular coats, at the plasma membrane, or both (Dale, 1983). An obvious acellular structure for sperm entry is the micropyle, found in the chorion of fish, squid and insect eggs. This narrow passage in the chorion physically restricts the number of spermatozoa reaching the egg surface. In the mollusc Unio, localization of sperm receptors on the vitelline coat to the vegetal hemisphere restricts sperm entry to this pole (Focarelli etal., 1988), while in the anuran Discoglossus, specializations of the jelly layer and vitelline coat contribute to ensuring that sperm entry exclusively occurs at a restricted area of the animal pole (Campanella et al., 1990). An additional factor limiting the entry of spermatozoa into eggs is the organization of the plasma membrane. In 1905, Conklin showed that sperm—egg fusion in ascidians is limited to an area of 30° at the vegetal pole. In many mammalian eggs, the plasma © Oxford University Press
Fig. 1. Scanning electron micrograph of the topographical distribution of plasma membrane microvilli in the unfertilized human oocyte in metaphase D. a A few corona cells (CC) and a piece of the zona pellucida (ZP) were left attached to the equatorial region of the oocyte for orientation. The polar body, lost during dissection, was located at 3 o'clock, b A higher magnification of the surface microvilli at that pole.
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L.Santdbj el al.
1980a,b; Phillips and Shalgi, 1982). In the mouse, rat and hamster, spermatozoa do not normally fuse with the microvillusfree area. Transmission electron microscopy studies have revealed the association of meiotic chromosomes and the formation or absence of the microvillus-free area (Longo and Chen, 1985; Pickering et al., 1985). A functional test for regionalization of the plasma membrane of the mammalian egg is localized microsurgery of the zona pellucida, which favours sperm entry at predetermined sites. Talansky et al. (1991) have confirmed, using this technique, that sperm entry in mouse and hamster eggs does not normally occur at the site of the first polar body. The purpose of the present study was to make a preliminary assessment of polarity in the human oocyte plasma membrane, using scanning electron microscopy and regionalized zona opening.
Materials and methods Source of oocytes for electron microscopy Spare oocytes were obtained at two different centres from consenting patients undergoing in-vitro fertilization (TVF) for infertility treatment. Follicular stimulation was performed using gonadotrophin administration (Dale et al., 1991). Oocytes were cultured in Earle's salt solution or human tubal fluid supplemented
with either 10% heat-inactivated human serum or 6% human serum albumin (HSA, Plasmanate; Cutter Biological, Miles Inc., Indiana, USA) and maintained at 37°C in a gas mixture of 5% COj in air (Cohen et al., 1985, 1991; Quinn et al., 1985). Only oocytes with an extruded first polar body and apparently normal morphology were used. Four different groups of oocytes were processed for scanning electron microscopy. The first group of 16 zona pellucida-intact, unfertilized oocytes were either fixed within 4 h following oocyte retrieval (Fertility Centre, Clinica Posillipo, Naples, Italy) or 12-15 h following initial insemination (The Centre for Reproduction and Infertility, Cornell University, New York USA). Excess corona cells were removed with hypodermic needles or using a narrow-bore glass pipette following the initial culture period. The zonae of oocytes of similar age from a second group (n = 11) were first partially opened by micromanipulating them with acidic Tyrode's solution (Gordon and Talanksy, 1986). The zona-free eggs were washed free from the acidic solution using regular culture medium. The third group of oocytes (n = 6) were zona-intact and fixed 96 h following collection in order to study the effect of ageing on membrane morphology. The fourth group (n = 7) had been micromanipulated for subzonal insertion 6 - 8 h following egg collection (Cohen et al., 1991). Two to four spermatozoa were introduced into the perivitelline space while the oocytes were maintained in a solution containing 0.1 M
Fig. 2. Scanning electron micrograph of the plasma membrane topography of an unfertilized human oocyte 14 h after insemination, a and b show the smooth polar body at 1 o'clock and the underlying microvilli; c and d show a similar topography at the opposite pole. Note there is no difference in morphology between the two poles. 1000
The human oocyte plasma membrane
sucrose. Sucrose was removed by serial washing and the oocytes were incubated in a sperm-free droplet of culture medium. These sucrose-exposed oocytes were fixed 12-15 h post-insemination. Electron microscopy The zona pellucida was removed using fine steel needles under a dissecting microscope. Oocytes were fixed in 3% gluteraJdehyde in 0.1 M sodium cacodylate buffer (pH = 7.2) for 1 — 2 h at room temperature and post-fixed in 1% osmium tetroxide. Following dehydration, samples were observed in a Philips 505 scanning electron microscope, or embedded in Epon 812, sectioned and studied in a Philips 400 transmission electron microscope.
seen extending from the oocyte surface to the polar body, an example of which is shown in Figure 2b. Figure 2c and d shows that the surface at the opposite hemisphere was covered by microvilli of similar size and frequency. In cases where die zona pellucida was removed using acidic Tyrode's solution, the oocyte surface was not homogeneously covered by microvilli (Figure 3a). Smooth areas of ^ 2 0 fim in diameter were consistendy seen over the oocyte surface although die general appearance of the microvilli was similar to diat of the control oocytes. A similar appearance was noted in aged oocytes, i.e. oocytes fixed ^ 4 days following in-vitro cultivation (Figure 3b). In the last group of oocytes used for subzonal sperm
Regionalized zona opening by micromanipulation The effect of introducing gaps in different regions of the zona pellucida in conjunction with subsequent insemination was evaluated on oocytes from 10 patients who consented to have partial zona dissection (PZD). The male partners of the patients had severely abnormal semen analyses and their spermatozoa failed to fertilize eggs in previous IVF attempts (Cohen et al., 1991). The experiments closely followed the procedures oudined by similar studies performed in the mouse and hamster (Talansky et al., 1991). At 4—7 h post-retrieval, oocytes were exposed to 0.1% hyaluronidase (sheep testes Type III, Sigma, St Louis, MO, USA) in phosphate-buffered saline (PBS) supplemented with pyruvate, penicillin and either 10% maternal serum or 6% HSA. Excess corona cells were removed with a narrow-bore glass pipette. Mature oocytes were selected, washed four times in culture medium and divided into two groups. The zona of the first group of oocytes was opened near the first polar body and in the second group, zonae were micromanipulated in a region opposite the first polar body. A total of 55 oocytes were thus micromanipulated. Partial zona dissection was performed as previously described (Cohen etal., 1989). Manipulated oocytes were washed through several microdrops of sucrose-free culture medium, and inseminated in 50-100 jtl microdrops at a concentration of 0.5-2 x 105 spermatozoa/ml. The oocytes were examined for the presence of pronuclei and polar bodies following 12-15 h of incubation.
Results Electron microscope studies The plasma membrane of the unfertilized metaphase II human oocytes was arranged in microvilli of 1 —3 fim in length. The uninseminated oocyte in Figure la, dissected in a 3% solution of gluteraldehyde 4 h after retrieval, showed the microvilli to be homogeneously distributed globally, while at the higher magnification it was seen that the individual microvilli were tubular and evenly spaced (Figure lb). Figure 2 shows several views of an oocyte 14 h after insemination that failed to fertilize. In Figure 2a and b the polar body was seen at 1 o'clock as a relatively smooth, microvillus-free sphere of about 10 /im in diameter. The oocyte surface below and around the polar body was covered by microvilli and characteristically long microvilli were
Fig. 3. Microvillus-free areas were consistently observed in human oocytes where the zona pellucida was removed with a acid Tyrode's, or b in oocytes cultured for 4 days before fixation. 1001
L.Santella et al.
insertion, the surface was often altered, with small round bodies and laterally extended whorl-like microvilli intermingled with the tubular microvilli (Figure 4a). At the level of the transmission electron microscope, these oocytes showed evidence of partial cortical granule exocytosis (Figure 4b). Regionalized micromanipulation of the zona pellucida Fertilization data relating to the site of PZD with respect to the polar body are presented in Figure 5. Fertilization rates were 48% in oocytes whose zonae were opened near the first polar body and 43% in oocytes micromanipulated at the opposite
hemisphere, suggesting that sperm-oocyte fusion may have occurred in both hemispheres. Since spermatozoa were only observed in the perivitelline space in 13% of PZD oocytes (from a separate control group of 77 unfertilized human PZD oocytes), it is unlikely that spermatozoa travelled laterally sub-zonally to fuse at a site distant from the gap. This was also confirmed in studies involving hamster and mouse oocytes (Talansky et al., 1991). A significantly higher rate of polyspermy resulted after PZD was performed near the polar body {P < 0.05; chi-square test). This may have been due to a local expansion of the perivitelline space when micromanipulation was performed at the site of the polar body, resulting in an accumulation of spermatozoa. Discussion
'r.-~r-'
u.
Using scanning electron microscopy, we have shown that the plasma membrane of the metaphase II human oocyte is an apparently homogeneous structure arranged in evenly spaced, short microvilli, which is not dissimilar to the surface of the regulative sea urchin oocyte (Hagstrom and Lonning, 1976; Schatten and Mazia, 1976; Dale et al., 1989). In most animal species studied to date, the frequency, shape and length of plasma membrane microvilli in the sperm-receptive oocyte appear to be similar, irrespective of the meiotic stage at ovulation. In contrast to other mammals (Johnson et al, 1975; Phillips and Shalgi, 1982; Shalgi and Phillips, 1980a,b), there is no microvillus-free area overlying the metaphase spindle in the human oocyte, although such areas are often present in aged oocytes and those exposed to low pH. This may in part explain the reduced developmental capacity of human embryos fertilized following zona drilling (Gordon et al., 1988; Maker and Cohen, 1989). The present observations extend suggestions of other workers regarding major differences in actin distribution, spindle orientation and polar body extrusion between the mouse and the human oocyte (Pickering et al., 1988). We have no direct morphological evidence to show that spermatozoa may fuse with both hemispheres of the human oocyte plasma membrane. However, our experiments with regionalized zona opening suggest that there is no preferential pole for sperm
Site of Zona Micromanipulation
Incidence of Fertilization Monospemuc
6/27 (22%)
12/28 (43%) Fig. 4. Scanning electron micrograph of the plasma membrane of human oocytes prepared for subzonal sperm insertion by osmotic shrinkage in sucrose showing that it was altered, with small round bodies and laterally extended whorl-like microvilli intermingled with the tubular microvilli (a). Sections of the same plasma membrane at the level of the transmission electron microscope showed evidence of partial cortical granule exocytosis (b, arrows).
1002
Polyspennic
Total
7/27 (26%)
13/27 (48%)
0/28 (0%)
12/28 (43%)
Fig. 5. Fertilization data relating the site of partial zona opening to the polar body in a trial using oocytes from 10 patients.
The human oocyte plasna membrane entry in the human at the level of the plasma membrane. Further support for this lack of functional polarity in the human oocyte is the pattern of polymerized cortical actin that differs from other mammalian species (Pickering etai, 1988). In addition, spermatozoa do not normally fuse with the microvillus-free area of other mammalian oocytes (Talansky et al., 1991), and in the hamster, immature oocytes attain competence to fuse with spermatozoa simultaneously with the appearance of microvilli (Zuccotti etai, 1991). In oocytes with microvillus-free areas, such as the mouse and hamster, the metaphase plate is peripherally located under a thick layer of cortical actin. Male pronuclear fusion at this pole would be disadvantageous. The absence of such cortical organization and the centralized position of the human oocyte metaphase plate (Pickering etai., 1988) supports our observation that sperm fusion is not polarized in the human. However, as suggested for the regulative sea urchin oocyte, the fact that a spermatozoon may fuse anywhere over the surface does not exclude the possibility of preferential entry sites or 'hot spots', randomly located over the surface (Dale, 1987).
References Campanella,C, Talevi.R., Gualteri.R. and Andreucetti.P. (1990) The contribution of Disoglossus pictus fertilization in the study of amphibian sperm-egg interaction. In Dale,B. (ed.), Mechanism of Fertilization: Plants to Humans. Nato ASI Series H, Vol. 45, SpnngerVerlag, Heidelberg, p. 557. CohenJ., Edwards,R.G., Fehilly,C.B., Fishel.S.B., Hewitt,J., Purdy.J.M., Rowland,R.F., Steptoe,P.C. and Webster,J.B. (1985) In vitro fertilization: a treatment for male factor infertility. Fertil. Sterii, 43, 422-433. Cohen,J., Malter.H., Wright,G., Kort.H., Massey,J. and Mitchell,D. (1989) Partial zona dissection of human oocytes when failure of zona penetration is anticipated. Hum. Reprod., 4, 435-442. Cohen,J., Talansky,B.E., Malter,H., Alikani,M., Adler.A., Reing,A., Berekley.A., Graf,M., Davis,O., Lm,H., Bedford,J.M. and Rosenwaks,Z. (1991) Microsurgical fertilization and teratozoospermia. Hum. Reprod., 6, 118-123. Conklin.E.G. (1905) Organization and cell lineage of the ascidian egg. J. Natl. Acad. Sci. Phil., 13, 1-28. Dale,B. (1983) Fertilization in Animals. Studies in Biology No. 157. Edward Arnold, London. Dale,B. (1987) Mechanisms of fertilization. Nature, 325, 762-763. Dale.B., Hagstrom,B. and Santella.L. (1989) Partially fertilized sea urchin eggs: an electrophysiological and morphological study. Dev. Growth Differ., 31, 165-170. Dale.B., Gualtieri,R., Talevi,R., Tosti.E., Santella,L. and Elder,K. (1991) Intercellular communication in the early human embryo. Mol. Reprod. Dev., 29, 22-28. FocarelIi,R., Renieri,T. and Rosati.F. (1988) Polarized site of sperm entry in the egg of a freshwater bivalve, Unto elongatus. Dev. Biol., 127, 443-451. Gordon,J.W. and Talansky,B.E. (1986) Assisted fertilization by zona drilling: a mouse model for correction of oligospermia. J. Exp. Zool., 239, 347-354. Gordon.J.W., Grunfeld,L., Garrisi.G.J., Talansky,B.E., Richards,C. and Laufer,N. (1988) Fertilization of human oocytes by sperm from infertile males after zona drilling. Fertil. Sterii., 50, 68-73. Hagstrom,B. and Lfinmng.S. (1976) Scanning electron microscope studies of the surface of sea urchin eggs. Protoplasma, 87, 281-290. Johnson,M.H., Eager,D. and Muggleton-Harris.A. (1975) Mosaicism
in organization of concanavalin A receptors on surface membrane of mouse eggs. Nature, 257, 321—322. Longo.F.J. and Chen,D.Y. (1985) Development of cortical polarity in mouse eggs: involvement of the meiotic apparatus. Dev. Biol., 107, 382-394. Malter,H.E. and Cohen.J. (1989) Partial zona dissection of the human oocyte: a nontraumatic method using rnicromanipulation to assist zona pellucida penetration. Fertil. Sterii., 51, 139-148. Monroy,A. (1965) Chemistry and Physiology of Fertilization. Holt, Rheinhart and Winston, New York. Philips,D.M. and Shalgi,R. (1982) Sperm penetration into rat ova fertilized in vivo. J. Exp. Biol., 221, 373-378. Pickering.S.J., Johnson,M.H., Braude,P.R. and Houliston,E. (1988) Cytoskeletal organization in fresh, aged and spontaneously activated human oocytes. Hum. Reprod., 3, 978-989. Quinn.P., Warnes.G.M., KeriiU.F. and Kirby.C. (1985) Culture factors affecting the success rate of IVF and embryo transfer. Ann. N. Y. Acad. Sci., 442, 195. Schatten,G. and Mazia.D. (1976) The penetration of the spermatozoon through the sea urchin egg at fertilization. Exp. Cell Res., 98, 325-337. Shalgi,R. and Phillips,D.M. (1980a) Mechanisms of in vitro fertilization in the hamster. Biol. Reprod., 23, 443-444. Shalgi.R.and Phillips,D.M. (1980b) Mechanisms of sperm entry in cycling hamster. J. Ultrastruct. Res., 71, 154-161. Talansky,B.E., Malter.H.E. and Cohen.J. (1991) The preferential site for sperm-egg fusion in mammals. Mol. Reprod. Dev., 28, 183 —188. Zuccotti,M., Yanagimachi,R. and Yanagimachi.H. (1991) The ability of the hamster oolemma to fuse with spermatozoa: its acquisition during oogenesis and loss after fertilization. Development, 112, 143-152. Received on January 13, 1992; accepted on April 13, 1992
1003
Is the human oocyte plasma membrane polarized?
Luigia Santella1, Mina Alikani2, Beth E.Talansky2, Jacques Cohen2-3 and Brian Dale1 'Stazione Zoologica, Villa Communale I, 80121, Napoli, Italy and The Gamete and Embryo Research Laboratory, The Center for Reproductive Medicine and Infertility, Department of Obstetrics and Gynecology, The New York Hospital—Cornell University Medical College, PO Box 30, 1300 York Avenue, New York, NY 10021, USA
membrane is organized in regularly spaced, short microvilli, except for an area overlying the metaphase spindle, which is essentially flat (Johnson etal., 1975; Shalgi and Phillips,
2
3
To whom corrspondence should be addressed
Using scanning electron microscopy, we have shown that the plasma membrane of the human metaphase II oocyte is organized in evenly spaced, short microvilli of 1 - 3 fim in length. In contrast to other mammals studied to date, there is no microvillus-free area overlying the metaphase spindle and there were no other indications of polarization at this level of organization. Functional polarity of the plasma membrane, studied using localized microsurgery of the zona pelhicida followed by insemination, suggests that sperm fusion and entry in the human may occur anywhere over the oocyte surface. Aged oocytes and those exposed to acidic Tyrode's solution had surfaces which were not homogeneously covered by microvilli. Oocytes exposed to a sucrose solution and subzonally injected with spermatozoa showed evidence of partial cortical granule exocytosis. Key words: first polar body/regional micromanipulation/ microvillus-free areayactivationypartial cortical exocytosis
Introduction In many animals, sperm entry is localized to a specific region of the egg surface (Monroy, 1965). This regionalization may be found at the level of the extracellular coats, at the plasma membrane, or both (Dale, 1983). An obvious acellular structure for sperm entry is the micropyle, found in the chorion of fish, squid and insect eggs. This narrow passage in the chorion physically restricts the number of spermatozoa reaching the egg surface. In the mollusc Unio, localization of sperm receptors on the vitelline coat to the vegetal hemisphere restricts sperm entry to this pole (Focarelli etal., 1988), while in the anuran Discoglossus, specializations of the jelly layer and vitelline coat contribute to ensuring that sperm entry exclusively occurs at a restricted area of the animal pole (Campanella et al., 1990). An additional factor limiting the entry of spermatozoa into eggs is the organization of the plasma membrane. In 1905, Conklin showed that sperm—egg fusion in ascidians is limited to an area of 30° at the vegetal pole. In many mammalian eggs, the plasma © Oxford University Press
Fig. 1. Scanning electron micrograph of the topographical distribution of plasma membrane microvilli in the unfertilized human oocyte in metaphase D. a A few corona cells (CC) and a piece of the zona pellucida (ZP) were left attached to the equatorial region of the oocyte for orientation. The polar body, lost during dissection, was located at 3 o'clock, b A higher magnification of the surface microvilli at that pole.
999
L.Santdbj el al.
1980a,b; Phillips and Shalgi, 1982). In the mouse, rat and hamster, spermatozoa do not normally fuse with the microvillusfree area. Transmission electron microscopy studies have revealed the association of meiotic chromosomes and the formation or absence of the microvillus-free area (Longo and Chen, 1985; Pickering et al., 1985). A functional test for regionalization of the plasma membrane of the mammalian egg is localized microsurgery of the zona pellucida, which favours sperm entry at predetermined sites. Talansky et al. (1991) have confirmed, using this technique, that sperm entry in mouse and hamster eggs does not normally occur at the site of the first polar body. The purpose of the present study was to make a preliminary assessment of polarity in the human oocyte plasma membrane, using scanning electron microscopy and regionalized zona opening.
Materials and methods Source of oocytes for electron microscopy Spare oocytes were obtained at two different centres from consenting patients undergoing in-vitro fertilization (TVF) for infertility treatment. Follicular stimulation was performed using gonadotrophin administration (Dale et al., 1991). Oocytes were cultured in Earle's salt solution or human tubal fluid supplemented
with either 10% heat-inactivated human serum or 6% human serum albumin (HSA, Plasmanate; Cutter Biological, Miles Inc., Indiana, USA) and maintained at 37°C in a gas mixture of 5% COj in air (Cohen et al., 1985, 1991; Quinn et al., 1985). Only oocytes with an extruded first polar body and apparently normal morphology were used. Four different groups of oocytes were processed for scanning electron microscopy. The first group of 16 zona pellucida-intact, unfertilized oocytes were either fixed within 4 h following oocyte retrieval (Fertility Centre, Clinica Posillipo, Naples, Italy) or 12-15 h following initial insemination (The Centre for Reproduction and Infertility, Cornell University, New York USA). Excess corona cells were removed with hypodermic needles or using a narrow-bore glass pipette following the initial culture period. The zonae of oocytes of similar age from a second group (n = 11) were first partially opened by micromanipulating them with acidic Tyrode's solution (Gordon and Talanksy, 1986). The zona-free eggs were washed free from the acidic solution using regular culture medium. The third group of oocytes (n = 6) were zona-intact and fixed 96 h following collection in order to study the effect of ageing on membrane morphology. The fourth group (n = 7) had been micromanipulated for subzonal insertion 6 - 8 h following egg collection (Cohen et al., 1991). Two to four spermatozoa were introduced into the perivitelline space while the oocytes were maintained in a solution containing 0.1 M
Fig. 2. Scanning electron micrograph of the plasma membrane topography of an unfertilized human oocyte 14 h after insemination, a and b show the smooth polar body at 1 o'clock and the underlying microvilli; c and d show a similar topography at the opposite pole. Note there is no difference in morphology between the two poles. 1000
The human oocyte plasma membrane
sucrose. Sucrose was removed by serial washing and the oocytes were incubated in a sperm-free droplet of culture medium. These sucrose-exposed oocytes were fixed 12-15 h post-insemination. Electron microscopy The zona pellucida was removed using fine steel needles under a dissecting microscope. Oocytes were fixed in 3% gluteraJdehyde in 0.1 M sodium cacodylate buffer (pH = 7.2) for 1 — 2 h at room temperature and post-fixed in 1% osmium tetroxide. Following dehydration, samples were observed in a Philips 505 scanning electron microscope, or embedded in Epon 812, sectioned and studied in a Philips 400 transmission electron microscope.
seen extending from the oocyte surface to the polar body, an example of which is shown in Figure 2b. Figure 2c and d shows that the surface at the opposite hemisphere was covered by microvilli of similar size and frequency. In cases where die zona pellucida was removed using acidic Tyrode's solution, the oocyte surface was not homogeneously covered by microvilli (Figure 3a). Smooth areas of ^ 2 0 fim in diameter were consistendy seen over the oocyte surface although die general appearance of the microvilli was similar to diat of the control oocytes. A similar appearance was noted in aged oocytes, i.e. oocytes fixed ^ 4 days following in-vitro cultivation (Figure 3b). In the last group of oocytes used for subzonal sperm
Regionalized zona opening by micromanipulation The effect of introducing gaps in different regions of the zona pellucida in conjunction with subsequent insemination was evaluated on oocytes from 10 patients who consented to have partial zona dissection (PZD). The male partners of the patients had severely abnormal semen analyses and their spermatozoa failed to fertilize eggs in previous IVF attempts (Cohen et al., 1991). The experiments closely followed the procedures oudined by similar studies performed in the mouse and hamster (Talansky et al., 1991). At 4—7 h post-retrieval, oocytes were exposed to 0.1% hyaluronidase (sheep testes Type III, Sigma, St Louis, MO, USA) in phosphate-buffered saline (PBS) supplemented with pyruvate, penicillin and either 10% maternal serum or 6% HSA. Excess corona cells were removed with a narrow-bore glass pipette. Mature oocytes were selected, washed four times in culture medium and divided into two groups. The zona of the first group of oocytes was opened near the first polar body and in the second group, zonae were micromanipulated in a region opposite the first polar body. A total of 55 oocytes were thus micromanipulated. Partial zona dissection was performed as previously described (Cohen etal., 1989). Manipulated oocytes were washed through several microdrops of sucrose-free culture medium, and inseminated in 50-100 jtl microdrops at a concentration of 0.5-2 x 105 spermatozoa/ml. The oocytes were examined for the presence of pronuclei and polar bodies following 12-15 h of incubation.
Results Electron microscope studies The plasma membrane of the unfertilized metaphase II human oocytes was arranged in microvilli of 1 —3 fim in length. The uninseminated oocyte in Figure la, dissected in a 3% solution of gluteraldehyde 4 h after retrieval, showed the microvilli to be homogeneously distributed globally, while at the higher magnification it was seen that the individual microvilli were tubular and evenly spaced (Figure lb). Figure 2 shows several views of an oocyte 14 h after insemination that failed to fertilize. In Figure 2a and b the polar body was seen at 1 o'clock as a relatively smooth, microvillus-free sphere of about 10 /im in diameter. The oocyte surface below and around the polar body was covered by microvilli and characteristically long microvilli were
Fig. 3. Microvillus-free areas were consistently observed in human oocytes where the zona pellucida was removed with a acid Tyrode's, or b in oocytes cultured for 4 days before fixation. 1001
L.Santella et al.
insertion, the surface was often altered, with small round bodies and laterally extended whorl-like microvilli intermingled with the tubular microvilli (Figure 4a). At the level of the transmission electron microscope, these oocytes showed evidence of partial cortical granule exocytosis (Figure 4b). Regionalized micromanipulation of the zona pellucida Fertilization data relating to the site of PZD with respect to the polar body are presented in Figure 5. Fertilization rates were 48% in oocytes whose zonae were opened near the first polar body and 43% in oocytes micromanipulated at the opposite
hemisphere, suggesting that sperm-oocyte fusion may have occurred in both hemispheres. Since spermatozoa were only observed in the perivitelline space in 13% of PZD oocytes (from a separate control group of 77 unfertilized human PZD oocytes), it is unlikely that spermatozoa travelled laterally sub-zonally to fuse at a site distant from the gap. This was also confirmed in studies involving hamster and mouse oocytes (Talansky et al., 1991). A significantly higher rate of polyspermy resulted after PZD was performed near the polar body {P < 0.05; chi-square test). This may have been due to a local expansion of the perivitelline space when micromanipulation was performed at the site of the polar body, resulting in an accumulation of spermatozoa. Discussion
'r.-~r-'
u.
Using scanning electron microscopy, we have shown that the plasma membrane of the metaphase II human oocyte is an apparently homogeneous structure arranged in evenly spaced, short microvilli, which is not dissimilar to the surface of the regulative sea urchin oocyte (Hagstrom and Lonning, 1976; Schatten and Mazia, 1976; Dale et al., 1989). In most animal species studied to date, the frequency, shape and length of plasma membrane microvilli in the sperm-receptive oocyte appear to be similar, irrespective of the meiotic stage at ovulation. In contrast to other mammals (Johnson et al, 1975; Phillips and Shalgi, 1982; Shalgi and Phillips, 1980a,b), there is no microvillus-free area overlying the metaphase spindle in the human oocyte, although such areas are often present in aged oocytes and those exposed to low pH. This may in part explain the reduced developmental capacity of human embryos fertilized following zona drilling (Gordon et al., 1988; Maker and Cohen, 1989). The present observations extend suggestions of other workers regarding major differences in actin distribution, spindle orientation and polar body extrusion between the mouse and the human oocyte (Pickering et al., 1988). We have no direct morphological evidence to show that spermatozoa may fuse with both hemispheres of the human oocyte plasma membrane. However, our experiments with regionalized zona opening suggest that there is no preferential pole for sperm
Site of Zona Micromanipulation
Incidence of Fertilization Monospemuc
6/27 (22%)
12/28 (43%) Fig. 4. Scanning electron micrograph of the plasma membrane of human oocytes prepared for subzonal sperm insertion by osmotic shrinkage in sucrose showing that it was altered, with small round bodies and laterally extended whorl-like microvilli intermingled with the tubular microvilli (a). Sections of the same plasma membrane at the level of the transmission electron microscope showed evidence of partial cortical granule exocytosis (b, arrows).
1002
Polyspennic
Total
7/27 (26%)
13/27 (48%)
0/28 (0%)
12/28 (43%)
Fig. 5. Fertilization data relating the site of partial zona opening to the polar body in a trial using oocytes from 10 patients.
The human oocyte plasna membrane entry in the human at the level of the plasma membrane. Further support for this lack of functional polarity in the human oocyte is the pattern of polymerized cortical actin that differs from other mammalian species (Pickering etai, 1988). In addition, spermatozoa do not normally fuse with the microvillus-free area of other mammalian oocytes (Talansky et al., 1991), and in the hamster, immature oocytes attain competence to fuse with spermatozoa simultaneously with the appearance of microvilli (Zuccotti etai, 1991). In oocytes with microvillus-free areas, such as the mouse and hamster, the metaphase plate is peripherally located under a thick layer of cortical actin. Male pronuclear fusion at this pole would be disadvantageous. The absence of such cortical organization and the centralized position of the human oocyte metaphase plate (Pickering etai., 1988) supports our observation that sperm fusion is not polarized in the human. However, as suggested for the regulative sea urchin oocyte, the fact that a spermatozoon may fuse anywhere over the surface does not exclude the possibility of preferential entry sites or 'hot spots', randomly located over the surface (Dale, 1987).
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