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A new method for preparation of pure zonae pellucidae in large quantities from porcine ovaries
Journal of Reproductive Immunology, 3 (1981) 147-156 Elsevier/North-Holland Biomedical Press
147
A NEW METHOD F O R PREPARATION O F PURE ZONAE PELLUCIDAE IN LARGE QUANTITIES FROM PORCINE OVARIES
YOICHI NODA, TAKAHIDE MORI, ICHIRO TAKAI, HEIGO KOHDA and TOSHIO NISHIMURA
Department of Obstetrics and Gynaecology, School of Medicine, Kyoto University Sakyo-ku, Kyoto 606, Japan (Received 29 October 1980; accepted 30 December 1980)
A new method for preparation of large quantities of zona substance in pure form from porcine ovaries has been described. In addition to the previously reported techniques of glass wool treatment and sieving by saran meshes, the following three significant improvements have been introduced: disruption of ovaries by an electric machine equipped with two multi-nee.died disks, which resulted in a considerably accelerated recovery of follicular oocytes with a high yield; low-speed centrffugation at 170 Xg for 15 s was found to be an obligatory step to eliminate light particulate material from the crude oocyte suspension; use of 50% sucrose solution in discontinuous density gradient centrifugation permitted complete separation of homogeneous samples at two stages of the f'mal preparation of zonaencased oocytes or of oocyte-free zonae. Microscopic examination revealed no contaminating components in the zona preparation. With this method 93 mg of lyophllized zona preparation were obtained from 24 553 porcine ovaries. Analysis of a solubilized zona substance by Sephaeryl S-200 column chromatography showed the presence of two major glycoproteins which could not be separated completely from each other. By analysis of the two components with SDS-PAGE, only a single, but broad, band of glycoprotein was found, indicating the successful isolation of a major component(s) from porcine zonae.
INTRODUCTION In addition to its importance for an understanding o f the process o f fertilization, the immunological properties o f the zona pellucida have recently attracted much attention in relation to the potential o f this membrane as a target for human immunocontraception (Shivers, 1977). The major immunologic criteria for this purpose seem fulfilled since specificity o f the zona antigen(s) has been established in mice (Tsunoda, 1977; Gwatkin et al., 1977), pigs (Sacco et al., 1977; Palm et al., 1979) and humans (Takai et al., 1981), using cumulus-free ova or isolated zonae pellucidae as immunogen, and antibodies to this structure have been shown to block fertilization in rodents b o t h in vivo and in vitro (Jilek et al., 1975; Tsunoda et al., 1976). Porcine zonae appear to be the best practical source o f the zona antigen(s) because o f ample availability as well as well documented cross-reactivity in antigenicity between human and porcine zonae (Sacco, 1977; Shivers et al., 1977; Takai et al., 1980). Despite earlier investigations using small quantities o f porcine zona pellucida (Sacco et al., 1977; Menino et al., 1979), the chemical and immunochemical nature o f the zona substance(s) are poorly understood, undoubtedly due to the limited availability o f the material. It is
0165-0378/81[0000-0000/$02.50 © Elsevier/North-Holland Biomedical Press
148 mandatory to obtain large quantities o f porcine zona substance(s) not only for analytical but for also preparative purposes. We have developed a satisfactory method to meet these purposes in terms of quantity as well as quality, a method that is distinct to a considerable extent from that recently reported by Dunbar and her colleagues (1980). MATERIALS AND METHODS
Isolation and collection or follicular oocytes Porcine ovaries were frozen immediately on removal at a local slaughterhouse, transported to the laboratory and kept at - 2 0 ° C until processed. Frozen ovaries were thawed at room temperature within 2 h by immersion under running water, and then transferred to 0.85% NaC1 solution containing 100 mM sodium phosphate, 0.01% sodium citrate and 0.01% sodium azide, pH 7.2 (referred to as PBS). Approximately 1000 to 2000 ovaries were processed at a time and all the isolation procedures were carried out at 0°C unless stated otherwise. For the ouroose of follicle disruption on a large scale, we invented a
Fig. 1. The follicle-disrupting machine used in the present study. A plastic girdle (20 cm diameter × 10 cm height) is set around the lower disk on the supporting stage to prevent splashing of follicular fluid and oocytes.
149 machine (YN type of follicle disruptor) with a pair of lead disks equipped with multiple needles on the surfaces facing each other (Fig. 1). While rotating at 200 rev./rnin by means of an electric motor fixed on the top of an axis, the upper disk moves down towards the lower disk fLxed on the supporting stage, thereby pressing and crushing the ovaries between the two disks. Treatment for 15 s was sufficient to disrupt all the follicles of 30 ovaries at a time. The oocytes, together with follicular fluid, tissue debris and granulosa cell masses, were recovered in a vessel placed under the lower disk through several openings set around the lower disk. After removing the ovarian debris, retained oocytes on the lower disk were washed down with 50 ml PBS and combined with the first pool of oocytes (referred to as crude oocyte suspension). 5 - 8 1 of crude oocyte suspension were obtained from 1000 to 2000 ovaries. The glass wool treatment described by Oikawa (1978) was followed for cleaning-up the crude oocyte suspension. The combined crude oocyte suspension was stirred by hand using a palmar-sized mass of non-activated glass wool for 30 s and the wet mass of glass wool was wrung to release the oocytes. Examination under a dissecting microscope revealed no detectable amount of oocytes in the glass wool. Ten times repetition of the glass wool treatment reduced the contaminants to a considerable but not to a satisfactory extent. Two steps of sieving procedures were carried out according to our original description (Mori et al., 1978), with modifications of pore sizes for further purification of the oocyte population. For the first step, the crude oocyte suspension was filtered through a sheet of saran net (70 X 70 cm) with a mesh of 250/am. Large pieces of tissue debris and aggregated cumulus ceils which had not been eliminated by the glass wool treatment were retained on the net. The filtrates were then passed through another saran net with a mesh
Fig. 2. Microscopic appearance of the oocyte preparation. Almost homogeneous populations of follicular oocytes are obtained with no contaminating cumulus cells, tissue debris or fragmented glass wool (X40).
150 of 110 /am to remove the substances smaller than the mesh size of the second net, for example erythrocytes and single cumulus cells. The oocytes retained on the second net were washed down into a glass beaker with PBS (referred to as oocyte suspension), then subjected to further glass wool treatment, after which the appearance o f the oocyte suspension became dearer and the presence o f oocytes could be seen as homogeneously scattering particulate material. The oocyte suspension was then freed from light contaminating components of cellular and subcellular sizes by low-speed centrifugation at 170 Xg for 15 s. This centrifugation procedure was repeated several times until the supernatant became clear. 20 ml of oocyte suspension were then overlayered on 30 ml of 50% sucrose solution (50% sucrose (w/v) in PBS) and centrifuged at 1600 Xg for 30 min. A clear oocyte layer was retained in the interphase between the sucrose and PBS layers, while relatively heavier contaminants precipitated at the b o t t o m of the sucrose layer. The recovered oocytes were washed with PBS several times (referred to as oocyte preparation) (Fig.2).
Fig. 3. Separation of porcine zonae by discontinuous density gradient centrifugation. 20 ml of homogenized oocyte suspension in PBS was overlayered on 30 ml of 50% sucrose solution and centrifuged at 1600 × g for 30 rain. A pure population of zonae precipitated at the bottom of the sucrose solution as a translucent layer (A), while a layer of amorphous particulate material and of fat granules concentrated at the interphase between sucrose and PBS layers (B), and on the top of PBS (C), respectively.
151
Preparation of oocyte-free zonae 1 ml of packed oocytes suspended in 3 ml PBS were gently homogenized by vertical strokes of the pestle 5 times in a glass homogenizer. The homogenate was overlayered on 30 ml of 50% sucrose solution and centrifuged at 1600 × g for 30 min. Oocyte-free zonae were clearly separated at the bottom of the tube as a translucent precipitate while visible particulate material and fat granules were concentrated at the interphase between the sucrose and PBS layers and on the surface of the PBS layer, respectively (Fig. 3). The recovered oocyte-free zonae were washed with several changes of PBS. Microscopically, the zona preparation consisted mostly of intact, but partially of fragmented pieces of zonae with some zonae retaining fat granules inside. The oocyte-free zonae were then resuspended in 10 times their volume of PBS and agitated vigorously for about 5 minutes by spurting the suspension from a Pasteur pipette to free the fat granules, then washed again several times with PBS by the low-speed centrifugation method (referred to as zona preparation) (Fig. 4). The zona preparation was dialysed against 4 1 of distilled water overnight, lyophilized and stored at -20°C until use. Solubilization of lyophilized zona preparation 20 mg of lyophilized zona preparation were incubated in 0.1 M Tris-HC1 buffer solution (80 ml, pH 8.6) containing 6 M urea and 0.02% EDTA (referred to as solubilization buffer) for 2 h at 60°C (Cholewa-Stewart, 1972) under nitrogen insufflation. Incubation was continued for an additional 30 min in the presence of 0.75 M ~-mercaptoethanol
Fig. 4. Microscopicappearance of the zona preparation, indicating an homogeneouspopulation of uncontaminated intact or disrupted zonae (× 100).
152 (Inoue et al., 1974) to achieve complete dissolution of the zonae. The mixture was carboxymethylated in the presence o f iodoacetic acid sodium salt at room temperature for 30 min in the dark; the amount of the reagent to be added was 3/4 mole equivalent to that of ~mercaptoethanol added to the solubilization buffer. The reaction mixture was then subjected to dialysis against 41 o f solubilization buffer overnight at 4°C in the dark. No detectable amounts of precipitate appeared at the end o f dialysis. The dialysate was reduced in volume to 3.5 ml using diaflo membrane (PM-10), then centrifuged at 12000 X g for 30 min and 19.95 mg protein were recovered in the supernatant (referred to as solubilized zona preparation).
Sephacryl S-200 column chromatography A Sephacryl S-200 (Pharmacia) column (1.5 X 33.5 cm) was equilibrated with the solubilization buffer. The elution o f the solubilized zona preparation was performed using the same buffer at room temperature at a flow rate of 4 ml/h, and was fractionated into 1.25 ml per tube. Elution profiles o f protein and neutral sugar were followed by absorbance at 280 nm and orcinol-sulfuric acid method (Francois et al., 1962), respectively. Protein was determined according to the method o f Lowry et al. (1951) using bovine serum albumin as a standard.
Polyacrylamide gel electrophoresis (PAGE) and densitometry Sodium dodecyl sulfate (SDS)-PAGE was performed essentially according to the TABLE 1 Recovery efficiency of follicular oocytes from porcine ovaries Expedmentno.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 Total
No. of ovaries used
Volume of packed oocytes recovered a (~l)
900 1 113 1 600 1 030 2030 2 200 2 000 2030 1 960 2 100 1 920 2060 2010 1 600
200 300 400 200 300 700 300 200 700 800 800 500 500 700
24 553
6 600
Yield of packed oocytes per 100 ovaries (ul) 22.2 27.0 25.0 19.4 14.8 31.8 15.0 9.9 35.7 38.1 41.7 24.3 24.9 43.8 26.7 b
a 100 t~l was estimated to correspond to approximately 10 000 oocytes by counting with a hemoeytometer. b Mean yield of packed oocytes per 100 ovaries.
153
method of Weber et al. (1972), employing 5.5, 6.0 and 7.0% gels. Gels were stained with Coomassie Brilliant Blue (R-250) or periodic acid-Schiff (Fairbanks et al., 1971). Densitometry was performed by a Densitron Model PAN (Jookoo Co. Ltd., Tokyo). RESULTS
Isolation and collection or follicular oocytes and oocyte-free zonae Following the above described procedures 6.6 ml of packed oocytes and a total of 93 mg lyophilized zona preparation were obtained from 24553 porcine ovaries through experiments repeated 14 times using 900 to 2200 ovaries at a time (Table 1). Microscopic examination of the oocyte preparation revealed a homogeneous oocyte population with no impurities (Fig. 2). The average diameter of the oocyte was 148.2 + 7.9/Jan (n = 30) inclusive of the zona, and the average width of the zona was 16.1 + 2.6 pm (n =
2.0
15
A oA_
Ii o
Lo. 0 3
0.5 0 25
il
~
25
50
75
Fraction Number (1.25ml/tube) Fig. 5. Elution profile of the solubflized zona substance on Sephacryl S-200 column chromatography. The sample was eluted by 0.1 M Tns-BCl buffer, pH 8.6, Ln the presence o f 6 M urea and 0.02% EDTA, at a flow rate of 4 ml/h with a be(] volume o f 60 nd. • e, absorbancc at 280 ran; o - - - % absorbance at 490 nm (orcinol-sulfuric acid reaction). The two peaks o f the solid line at traction Nos. 24 and 26 correspond to the two peaks of the dotted 1Lne,suggesting that the two components were glycoproteins.
154 30). Although considerable differences in size and shape were observed between zonae, no appreciable amount of contaminants was found in the zona preparation (Fig. 4). Based on the microscopic findings, the purity of the oocyte and zona preparations was judged to be satisfactory.
Chromatographic and gel eleetrophoretic patterns o f the solubilized zona preparation The elution profile of the solubilized zona preparation from Sephacryl S-200 column chromatography gave two major peaks of 280 nm absorbance at the fraction Nos. 24 and 26 (Fig. 5). Intensity of the neutral sugar reaction coincided with the same fraction numbers. The position of the two peaks was between those of blue dextran and bovine serum albumin which corresponded to the fraction Nos. 22 and 25, respectively. The fractions from 23 to 29 were combined and 16.8 mg protein were recovered, being 84% of the total amount (19.95 mg) applied. Electrophoresis of 30/~g protein from the combined fractions, when performed in 6.0% gel, gave a single broad band with positive protein and carbohydrate stainings, showing a Rm value of 0.49. The molecular weight of the glycoprotein was calibrated to be 64000 as compared to marker proteins. No other faint band was detected (Figs. 6A, B). This pattern was reproduced regardless of gel concentrations (5.5% or 7.0%) or the amount of protein applied (15 or 50/ag). In the companion gel, in which oocytederived substance was run, multiple bands were observed. However, none of them showed positive carbohydrate staining (data not shown).
E3
A
a
b
ji ii
I
i ¸
.2
.i
i
i
iJ
MOBILITY
Fig. 6. A. SDS-polyacrylamide gel electrophoresis of zona substance. The gels were stained with Coomassie Blue for protein (a) and with periodic acid-Schiff for neutral sugar (b). 50 #g of zona substance were applied to the gel. B. A pattern of densitometry scanned at 600 nm. 30 ~g of zona substance were applied to the gel.
155 DISCUSSION The method developed in the present study offers a useful tool for obtaining large quantities of zona material of high purity from porcine ovaries and could well be extended to other mammalian species. The use of machinery with multineedled plates instead of manual disruption of follicles increased by approximately 2.5 the number of porcine ovaries that could be treated per unit time, although the number ofoocytes harvested from one ovary seemed relatively larger by the manual than by the mechanical procedure. The mechanical method made it possible to treat a large number of ovaries in a short time, for example only 4 h for 2000 ovaries treated by two persons. The second approach unique to the present method is the use of low-speed centrifugation (170 ×g) for a short period of 15 s. The glass wool treatment is particularly effective in eliminating larger pieces of tissue debris and aggregated cumulus cell masses. By contrast, the low speed centrifugation was useful to remove light particulate material. The combined use of these two complementary procedures thus provides an essential step in the purification of the oocyte suspension. The third, and perhaps the most important, procedure is the sucrose density gradient centrifugation employed at two stages as a final means for purification either of the oocyte or of the zona preparation. The procedure is especially valuable in the purification of the oocyte preparation, because relatively heavier contaminants in the oocyte suspension such as tissue debris, aggregated cell masses and broken fragments of glass wool fibers were entirely eliminated. On the other hand, manual collection of cumulus-free oocytes by micropipetting is preferable for human ovaries (Mori et al., 1979), where it is important to recover as many oocytes as possible from each ovary. However, it is evident that such a manual procedure is no longer applicable for treating large quantities of ovaries. Choice of method for solubilization of zona substance requires careful consideration of subsequent analytical procedures. According to the reported methods, zona preparations can be solubilized under several experimental conditions, such as 3 h incubation at 70°C in distilled water (Gwatkin et al., 1977) or in 0.1 M sodium borate, pH 10.0 (Dunbar et al., 1980). We have tried using these two methods but have found 0.1 M Tris-HCl, pH 8.6, containing 6 M urea and 0.75 M/3-mercaptoethanol most suitable as a solubilizing vehicle for stabilizing the effect of the solution on the solubilized zona components because the two former solutions cannot prevent aggregation of solubilized zona components when the protein concentration is more than 1 mg/ml at 4°C. On analysis with SDS-PAGE only a single band of glycoprotein was produced, in agreement with the report by Sacco et al. (1977) but not with the report by Dunbar et al. (1980) claiming two glycoprotein bands. A major causative factor may be the difference in solubilization procedures. The elution profile of Sephacryl S-200 column chromatography showed two peaks of glycoproteins as the major components with no apparent peak of a minor component. On the other hand, only a single, but broad, band of glycoprotein was observed in any of the different experimental conditions employed in the SDS-polyacrylamide gel. This discrepancy in the number of glycoprotein components cannot be explained satisfactorily at the present time. However, these results suggest that porcine zonae are composed of simple molecular species and that the zona preparation obtained is of high quality. Further physicochemical, biochemical and immunological studies are in progress.
156 ACKNOWLEDGEMENTS This w o r k w a s supported in part f r o m The Ministry o f Health and Welfare, Japan and Fujiwara Memorial F o u n d a t i o n . REFERENCES Cholewa-Stewart, J. and Massaro, E.J. (1972) Thermally-induced dissolution of the murine zona peUucida. Biol. Reprod. 7,166-169. Dunbar, B.S., Wardrip, N.J. and Hedrick, J.L. (1980) Isolation, chemical properties and maeromolecular composition of zona pellucida from porcine oocytes. Biochemistry 19, 356-365. Fairbanks, G., Steck, T.L. and Wallach, D.F.H. (1971) Electrophoretic analysis of the major polypeptides of the human erythroeyte membrane. Biochemistry 10, 2606-2616. Francois, D., Marshall, R.D. and Neuberger, A. (1962) Carbohydrate in protein. Biochem. J. 8 3 , 3 3 5 341. Gwatkin, R.B.L., Williams, D.T. and Carlo, D.J. (1977) Immunization of mice with heat-solubilized hamster zonae: production of anti-zona antibody and inhibition of fertility. Fertil. Steril. 28, 871-877. Inoue, M. and Wolf, D.P. (1974) Solubility properties of the murine zona pellucida. Biol. Reprod. 10, 512-518. Jilek, F. and Pavlok, A. (1975) Antibodies against mouse ovaries and their effect on fertilization in vitro and in vivo in the mouse. J~ Reprod. Fert. 42,377-380. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem. 193,265-275. Menino, A.R. Jr. and Wright, R.W. Jr. (1979) Characterization of porcine oocyte zonae pellueidae by polyacrylamide gel,electrophoresis. Proc. Soe. Exp. Biol. Med. 160,449-452. Mori, T., Nishimoto, T., Kitagawa, M., Noda, Y., Nishimura, T. and Oikawa, T. (1978) Possible presence of autoantibodies to zona pellucida in infertile women. Experientia 34, 797-798. Mori, T., Nishimoto, T., Kohda, H., Takai, I., Nishimura, T. and Oikawa, T. (1979) A method for specific detection of autoantibodies to the zona pellucida in infertile women. Fertll. Steril. 32, 67 -72. Oikawa, T. (1978) A simple method for the isolation of a large number of ova from pig ovaries. Gamete Res. 1,265-267. Palm, V.S., Sacco, A.G., Syner, F.N. and Subramanian, M.G. (1979) Tissue specificity of protein zona pellucida antigen(s) tested by radioirnmunoassay. Biol. Reprod. 21,709-713. Sacco, A.G. (1977) Antigenic cross-reactivity between human and pig zona pellucida. Biol. Reprod. 16, 164-173. Sacco, A.G. and Palm, V.S. (1977) Heterolmmunization with isolated pig zonae peUucidae. J. Reprod. Fert. 51,165-168. Shivers, C.A. (1977) The zona pellucida as a possible target in immunocontraception. In Immunological Influence on Human Fertility (Boettcher, B. ed.), pp. 13-24. Academic Press, New York. Shivers, A.G. and Dunbar, B.S. (1977) Autoantibodies to zona pellucida: A possible cause for infertility in women. Science 197, 1082-1084. Takai, I., Mori, T., Noda, Y. and Nishlmura, T. (1981) Heteroimmunization with isolated human ova. J. Reprod. Fert. 61, 19-24. Tsunoda, Y. (1977) Inhibitory effect of anti-mouse egg serum on fertilization in vitro and in vivo in the mouse. J. Reprod. Fert. 50, 353-355. Tsunoda, Y. and Chang, M.C. (1976) Effect of anti-rat ovary antiserum on the fertilization of rat, mouse and hamster eggs in vivo and in vitro. Biol. Reprod. 14, 354-361. Weber, K., Pringle, J.R. and Osborn, M~ (1972) Measurement of molecular weights by electrophoresis on SDS-acrylamide gel. Methods Enzymol. 26, 3-27.
147
A NEW METHOD F O R PREPARATION O F PURE ZONAE PELLUCIDAE IN LARGE QUANTITIES FROM PORCINE OVARIES
YOICHI NODA, TAKAHIDE MORI, ICHIRO TAKAI, HEIGO KOHDA and TOSHIO NISHIMURA
Department of Obstetrics and Gynaecology, School of Medicine, Kyoto University Sakyo-ku, Kyoto 606, Japan (Received 29 October 1980; accepted 30 December 1980)
A new method for preparation of large quantities of zona substance in pure form from porcine ovaries has been described. In addition to the previously reported techniques of glass wool treatment and sieving by saran meshes, the following three significant improvements have been introduced: disruption of ovaries by an electric machine equipped with two multi-nee.died disks, which resulted in a considerably accelerated recovery of follicular oocytes with a high yield; low-speed centrffugation at 170 Xg for 15 s was found to be an obligatory step to eliminate light particulate material from the crude oocyte suspension; use of 50% sucrose solution in discontinuous density gradient centrifugation permitted complete separation of homogeneous samples at two stages of the f'mal preparation of zonaencased oocytes or of oocyte-free zonae. Microscopic examination revealed no contaminating components in the zona preparation. With this method 93 mg of lyophllized zona preparation were obtained from 24 553 porcine ovaries. Analysis of a solubilized zona substance by Sephaeryl S-200 column chromatography showed the presence of two major glycoproteins which could not be separated completely from each other. By analysis of the two components with SDS-PAGE, only a single, but broad, band of glycoprotein was found, indicating the successful isolation of a major component(s) from porcine zonae.
INTRODUCTION In addition to its importance for an understanding o f the process o f fertilization, the immunological properties o f the zona pellucida have recently attracted much attention in relation to the potential o f this membrane as a target for human immunocontraception (Shivers, 1977). The major immunologic criteria for this purpose seem fulfilled since specificity o f the zona antigen(s) has been established in mice (Tsunoda, 1977; Gwatkin et al., 1977), pigs (Sacco et al., 1977; Palm et al., 1979) and humans (Takai et al., 1981), using cumulus-free ova or isolated zonae pellucidae as immunogen, and antibodies to this structure have been shown to block fertilization in rodents b o t h in vivo and in vitro (Jilek et al., 1975; Tsunoda et al., 1976). Porcine zonae appear to be the best practical source o f the zona antigen(s) because o f ample availability as well as well documented cross-reactivity in antigenicity between human and porcine zonae (Sacco, 1977; Shivers et al., 1977; Takai et al., 1980). Despite earlier investigations using small quantities o f porcine zona pellucida (Sacco et al., 1977; Menino et al., 1979), the chemical and immunochemical nature o f the zona substance(s) are poorly understood, undoubtedly due to the limited availability o f the material. It is
0165-0378/81[0000-0000/$02.50 © Elsevier/North-Holland Biomedical Press
148 mandatory to obtain large quantities o f porcine zona substance(s) not only for analytical but for also preparative purposes. We have developed a satisfactory method to meet these purposes in terms of quantity as well as quality, a method that is distinct to a considerable extent from that recently reported by Dunbar and her colleagues (1980). MATERIALS AND METHODS
Isolation and collection or follicular oocytes Porcine ovaries were frozen immediately on removal at a local slaughterhouse, transported to the laboratory and kept at - 2 0 ° C until processed. Frozen ovaries were thawed at room temperature within 2 h by immersion under running water, and then transferred to 0.85% NaC1 solution containing 100 mM sodium phosphate, 0.01% sodium citrate and 0.01% sodium azide, pH 7.2 (referred to as PBS). Approximately 1000 to 2000 ovaries were processed at a time and all the isolation procedures were carried out at 0°C unless stated otherwise. For the ouroose of follicle disruption on a large scale, we invented a
Fig. 1. The follicle-disrupting machine used in the present study. A plastic girdle (20 cm diameter × 10 cm height) is set around the lower disk on the supporting stage to prevent splashing of follicular fluid and oocytes.
149 machine (YN type of follicle disruptor) with a pair of lead disks equipped with multiple needles on the surfaces facing each other (Fig. 1). While rotating at 200 rev./rnin by means of an electric motor fixed on the top of an axis, the upper disk moves down towards the lower disk fLxed on the supporting stage, thereby pressing and crushing the ovaries between the two disks. Treatment for 15 s was sufficient to disrupt all the follicles of 30 ovaries at a time. The oocytes, together with follicular fluid, tissue debris and granulosa cell masses, were recovered in a vessel placed under the lower disk through several openings set around the lower disk. After removing the ovarian debris, retained oocytes on the lower disk were washed down with 50 ml PBS and combined with the first pool of oocytes (referred to as crude oocyte suspension). 5 - 8 1 of crude oocyte suspension were obtained from 1000 to 2000 ovaries. The glass wool treatment described by Oikawa (1978) was followed for cleaning-up the crude oocyte suspension. The combined crude oocyte suspension was stirred by hand using a palmar-sized mass of non-activated glass wool for 30 s and the wet mass of glass wool was wrung to release the oocytes. Examination under a dissecting microscope revealed no detectable amount of oocytes in the glass wool. Ten times repetition of the glass wool treatment reduced the contaminants to a considerable but not to a satisfactory extent. Two steps of sieving procedures were carried out according to our original description (Mori et al., 1978), with modifications of pore sizes for further purification of the oocyte population. For the first step, the crude oocyte suspension was filtered through a sheet of saran net (70 X 70 cm) with a mesh of 250/am. Large pieces of tissue debris and aggregated cumulus ceils which had not been eliminated by the glass wool treatment were retained on the net. The filtrates were then passed through another saran net with a mesh
Fig. 2. Microscopic appearance of the oocyte preparation. Almost homogeneous populations of follicular oocytes are obtained with no contaminating cumulus cells, tissue debris or fragmented glass wool (X40).
150 of 110 /am to remove the substances smaller than the mesh size of the second net, for example erythrocytes and single cumulus cells. The oocytes retained on the second net were washed down into a glass beaker with PBS (referred to as oocyte suspension), then subjected to further glass wool treatment, after which the appearance o f the oocyte suspension became dearer and the presence o f oocytes could be seen as homogeneously scattering particulate material. The oocyte suspension was then freed from light contaminating components of cellular and subcellular sizes by low-speed centrifugation at 170 Xg for 15 s. This centrifugation procedure was repeated several times until the supernatant became clear. 20 ml of oocyte suspension were then overlayered on 30 ml of 50% sucrose solution (50% sucrose (w/v) in PBS) and centrifuged at 1600 Xg for 30 min. A clear oocyte layer was retained in the interphase between the sucrose and PBS layers, while relatively heavier contaminants precipitated at the b o t t o m of the sucrose layer. The recovered oocytes were washed with PBS several times (referred to as oocyte preparation) (Fig.2).
Fig. 3. Separation of porcine zonae by discontinuous density gradient centrifugation. 20 ml of homogenized oocyte suspension in PBS was overlayered on 30 ml of 50% sucrose solution and centrifuged at 1600 × g for 30 rain. A pure population of zonae precipitated at the bottom of the sucrose solution as a translucent layer (A), while a layer of amorphous particulate material and of fat granules concentrated at the interphase between sucrose and PBS layers (B), and on the top of PBS (C), respectively.
151
Preparation of oocyte-free zonae 1 ml of packed oocytes suspended in 3 ml PBS were gently homogenized by vertical strokes of the pestle 5 times in a glass homogenizer. The homogenate was overlayered on 30 ml of 50% sucrose solution and centrifuged at 1600 × g for 30 min. Oocyte-free zonae were clearly separated at the bottom of the tube as a translucent precipitate while visible particulate material and fat granules were concentrated at the interphase between the sucrose and PBS layers and on the surface of the PBS layer, respectively (Fig. 3). The recovered oocyte-free zonae were washed with several changes of PBS. Microscopically, the zona preparation consisted mostly of intact, but partially of fragmented pieces of zonae with some zonae retaining fat granules inside. The oocyte-free zonae were then resuspended in 10 times their volume of PBS and agitated vigorously for about 5 minutes by spurting the suspension from a Pasteur pipette to free the fat granules, then washed again several times with PBS by the low-speed centrifugation method (referred to as zona preparation) (Fig. 4). The zona preparation was dialysed against 4 1 of distilled water overnight, lyophilized and stored at -20°C until use. Solubilization of lyophilized zona preparation 20 mg of lyophilized zona preparation were incubated in 0.1 M Tris-HC1 buffer solution (80 ml, pH 8.6) containing 6 M urea and 0.02% EDTA (referred to as solubilization buffer) for 2 h at 60°C (Cholewa-Stewart, 1972) under nitrogen insufflation. Incubation was continued for an additional 30 min in the presence of 0.75 M ~-mercaptoethanol
Fig. 4. Microscopicappearance of the zona preparation, indicating an homogeneouspopulation of uncontaminated intact or disrupted zonae (× 100).
152 (Inoue et al., 1974) to achieve complete dissolution of the zonae. The mixture was carboxymethylated in the presence o f iodoacetic acid sodium salt at room temperature for 30 min in the dark; the amount of the reagent to be added was 3/4 mole equivalent to that of ~mercaptoethanol added to the solubilization buffer. The reaction mixture was then subjected to dialysis against 41 o f solubilization buffer overnight at 4°C in the dark. No detectable amounts of precipitate appeared at the end o f dialysis. The dialysate was reduced in volume to 3.5 ml using diaflo membrane (PM-10), then centrifuged at 12000 X g for 30 min and 19.95 mg protein were recovered in the supernatant (referred to as solubilized zona preparation).
Sephacryl S-200 column chromatography A Sephacryl S-200 (Pharmacia) column (1.5 X 33.5 cm) was equilibrated with the solubilization buffer. The elution o f the solubilized zona preparation was performed using the same buffer at room temperature at a flow rate of 4 ml/h, and was fractionated into 1.25 ml per tube. Elution profiles o f protein and neutral sugar were followed by absorbance at 280 nm and orcinol-sulfuric acid method (Francois et al., 1962), respectively. Protein was determined according to the method o f Lowry et al. (1951) using bovine serum albumin as a standard.
Polyacrylamide gel electrophoresis (PAGE) and densitometry Sodium dodecyl sulfate (SDS)-PAGE was performed essentially according to the TABLE 1 Recovery efficiency of follicular oocytes from porcine ovaries Expedmentno.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 Total
No. of ovaries used
Volume of packed oocytes recovered a (~l)
900 1 113 1 600 1 030 2030 2 200 2 000 2030 1 960 2 100 1 920 2060 2010 1 600
200 300 400 200 300 700 300 200 700 800 800 500 500 700
24 553
6 600
Yield of packed oocytes per 100 ovaries (ul) 22.2 27.0 25.0 19.4 14.8 31.8 15.0 9.9 35.7 38.1 41.7 24.3 24.9 43.8 26.7 b
a 100 t~l was estimated to correspond to approximately 10 000 oocytes by counting with a hemoeytometer. b Mean yield of packed oocytes per 100 ovaries.
153
method of Weber et al. (1972), employing 5.5, 6.0 and 7.0% gels. Gels were stained with Coomassie Brilliant Blue (R-250) or periodic acid-Schiff (Fairbanks et al., 1971). Densitometry was performed by a Densitron Model PAN (Jookoo Co. Ltd., Tokyo). RESULTS
Isolation and collection or follicular oocytes and oocyte-free zonae Following the above described procedures 6.6 ml of packed oocytes and a total of 93 mg lyophilized zona preparation were obtained from 24553 porcine ovaries through experiments repeated 14 times using 900 to 2200 ovaries at a time (Table 1). Microscopic examination of the oocyte preparation revealed a homogeneous oocyte population with no impurities (Fig. 2). The average diameter of the oocyte was 148.2 + 7.9/Jan (n = 30) inclusive of the zona, and the average width of the zona was 16.1 + 2.6 pm (n =
2.0
15
A oA_
Ii o
Lo. 0 3
0.5 0 25
il
~
25
50
75
Fraction Number (1.25ml/tube) Fig. 5. Elution profile of the solubflized zona substance on Sephacryl S-200 column chromatography. The sample was eluted by 0.1 M Tns-BCl buffer, pH 8.6, Ln the presence o f 6 M urea and 0.02% EDTA, at a flow rate of 4 ml/h with a be(] volume o f 60 nd. • e, absorbancc at 280 ran; o - - - % absorbance at 490 nm (orcinol-sulfuric acid reaction). The two peaks o f the solid line at traction Nos. 24 and 26 correspond to the two peaks of the dotted 1Lne,suggesting that the two components were glycoproteins.
154 30). Although considerable differences in size and shape were observed between zonae, no appreciable amount of contaminants was found in the zona preparation (Fig. 4). Based on the microscopic findings, the purity of the oocyte and zona preparations was judged to be satisfactory.
Chromatographic and gel eleetrophoretic patterns o f the solubilized zona preparation The elution profile of the solubilized zona preparation from Sephacryl S-200 column chromatography gave two major peaks of 280 nm absorbance at the fraction Nos. 24 and 26 (Fig. 5). Intensity of the neutral sugar reaction coincided with the same fraction numbers. The position of the two peaks was between those of blue dextran and bovine serum albumin which corresponded to the fraction Nos. 22 and 25, respectively. The fractions from 23 to 29 were combined and 16.8 mg protein were recovered, being 84% of the total amount (19.95 mg) applied. Electrophoresis of 30/~g protein from the combined fractions, when performed in 6.0% gel, gave a single broad band with positive protein and carbohydrate stainings, showing a Rm value of 0.49. The molecular weight of the glycoprotein was calibrated to be 64000 as compared to marker proteins. No other faint band was detected (Figs. 6A, B). This pattern was reproduced regardless of gel concentrations (5.5% or 7.0%) or the amount of protein applied (15 or 50/ag). In the companion gel, in which oocytederived substance was run, multiple bands were observed. However, none of them showed positive carbohydrate staining (data not shown).
E3
A
a
b
ji ii
I
i ¸
.2
.i
i
i
iJ
MOBILITY
Fig. 6. A. SDS-polyacrylamide gel electrophoresis of zona substance. The gels were stained with Coomassie Blue for protein (a) and with periodic acid-Schiff for neutral sugar (b). 50 #g of zona substance were applied to the gel. B. A pattern of densitometry scanned at 600 nm. 30 ~g of zona substance were applied to the gel.
155 DISCUSSION The method developed in the present study offers a useful tool for obtaining large quantities of zona material of high purity from porcine ovaries and could well be extended to other mammalian species. The use of machinery with multineedled plates instead of manual disruption of follicles increased by approximately 2.5 the number of porcine ovaries that could be treated per unit time, although the number ofoocytes harvested from one ovary seemed relatively larger by the manual than by the mechanical procedure. The mechanical method made it possible to treat a large number of ovaries in a short time, for example only 4 h for 2000 ovaries treated by two persons. The second approach unique to the present method is the use of low-speed centrifugation (170 ×g) for a short period of 15 s. The glass wool treatment is particularly effective in eliminating larger pieces of tissue debris and aggregated cumulus cell masses. By contrast, the low speed centrifugation was useful to remove light particulate material. The combined use of these two complementary procedures thus provides an essential step in the purification of the oocyte suspension. The third, and perhaps the most important, procedure is the sucrose density gradient centrifugation employed at two stages as a final means for purification either of the oocyte or of the zona preparation. The procedure is especially valuable in the purification of the oocyte preparation, because relatively heavier contaminants in the oocyte suspension such as tissue debris, aggregated cell masses and broken fragments of glass wool fibers were entirely eliminated. On the other hand, manual collection of cumulus-free oocytes by micropipetting is preferable for human ovaries (Mori et al., 1979), where it is important to recover as many oocytes as possible from each ovary. However, it is evident that such a manual procedure is no longer applicable for treating large quantities of ovaries. Choice of method for solubilization of zona substance requires careful consideration of subsequent analytical procedures. According to the reported methods, zona preparations can be solubilized under several experimental conditions, such as 3 h incubation at 70°C in distilled water (Gwatkin et al., 1977) or in 0.1 M sodium borate, pH 10.0 (Dunbar et al., 1980). We have tried using these two methods but have found 0.1 M Tris-HCl, pH 8.6, containing 6 M urea and 0.75 M/3-mercaptoethanol most suitable as a solubilizing vehicle for stabilizing the effect of the solution on the solubilized zona components because the two former solutions cannot prevent aggregation of solubilized zona components when the protein concentration is more than 1 mg/ml at 4°C. On analysis with SDS-PAGE only a single band of glycoprotein was produced, in agreement with the report by Sacco et al. (1977) but not with the report by Dunbar et al. (1980) claiming two glycoprotein bands. A major causative factor may be the difference in solubilization procedures. The elution profile of Sephacryl S-200 column chromatography showed two peaks of glycoproteins as the major components with no apparent peak of a minor component. On the other hand, only a single, but broad, band of glycoprotein was observed in any of the different experimental conditions employed in the SDS-polyacrylamide gel. This discrepancy in the number of glycoprotein components cannot be explained satisfactorily at the present time. However, these results suggest that porcine zonae are composed of simple molecular species and that the zona preparation obtained is of high quality. Further physicochemical, biochemical and immunological studies are in progress.
156 ACKNOWLEDGEMENTS This w o r k w a s supported in part f r o m The Ministry o f Health and Welfare, Japan and Fujiwara Memorial F o u n d a t i o n . REFERENCES Cholewa-Stewart, J. and Massaro, E.J. (1972) Thermally-induced dissolution of the murine zona peUucida. Biol. Reprod. 7,166-169. Dunbar, B.S., Wardrip, N.J. and Hedrick, J.L. (1980) Isolation, chemical properties and maeromolecular composition of zona pellucida from porcine oocytes. Biochemistry 19, 356-365. Fairbanks, G., Steck, T.L. and Wallach, D.F.H. (1971) Electrophoretic analysis of the major polypeptides of the human erythroeyte membrane. Biochemistry 10, 2606-2616. Francois, D., Marshall, R.D. and Neuberger, A. (1962) Carbohydrate in protein. Biochem. J. 8 3 , 3 3 5 341. Gwatkin, R.B.L., Williams, D.T. and Carlo, D.J. (1977) Immunization of mice with heat-solubilized hamster zonae: production of anti-zona antibody and inhibition of fertility. Fertil. Steril. 28, 871-877. Inoue, M. and Wolf, D.P. (1974) Solubility properties of the murine zona pellucida. Biol. Reprod. 10, 512-518. Jilek, F. and Pavlok, A. (1975) Antibodies against mouse ovaries and their effect on fertilization in vitro and in vivo in the mouse. J~ Reprod. Fert. 42,377-380. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem. 193,265-275. Menino, A.R. Jr. and Wright, R.W. Jr. (1979) Characterization of porcine oocyte zonae pellueidae by polyacrylamide gel,electrophoresis. Proc. Soe. Exp. Biol. Med. 160,449-452. Mori, T., Nishimoto, T., Kitagawa, M., Noda, Y., Nishimura, T. and Oikawa, T. (1978) Possible presence of autoantibodies to zona pellucida in infertile women. Experientia 34, 797-798. Mori, T., Nishimoto, T., Kohda, H., Takai, I., Nishimura, T. and Oikawa, T. (1979) A method for specific detection of autoantibodies to the zona pellucida in infertile women. Fertll. Steril. 32, 67 -72. Oikawa, T. (1978) A simple method for the isolation of a large number of ova from pig ovaries. Gamete Res. 1,265-267. Palm, V.S., Sacco, A.G., Syner, F.N. and Subramanian, M.G. (1979) Tissue specificity of protein zona pellucida antigen(s) tested by radioirnmunoassay. Biol. Reprod. 21,709-713. Sacco, A.G. (1977) Antigenic cross-reactivity between human and pig zona pellucida. Biol. Reprod. 16, 164-173. Sacco, A.G. and Palm, V.S. (1977) Heterolmmunization with isolated pig zonae peUucidae. J. Reprod. Fert. 51,165-168. Shivers, C.A. (1977) The zona pellucida as a possible target in immunocontraception. In Immunological Influence on Human Fertility (Boettcher, B. ed.), pp. 13-24. Academic Press, New York. Shivers, A.G. and Dunbar, B.S. (1977) Autoantibodies to zona pellucida: A possible cause for infertility in women. Science 197, 1082-1084. Takai, I., Mori, T., Noda, Y. and Nishlmura, T. (1981) Heteroimmunization with isolated human ova. J. Reprod. Fert. 61, 19-24. Tsunoda, Y. (1977) Inhibitory effect of anti-mouse egg serum on fertilization in vitro and in vivo in the mouse. J. Reprod. Fert. 50, 353-355. Tsunoda, Y. and Chang, M.C. (1976) Effect of anti-rat ovary antiserum on the fertilization of rat, mouse and hamster eggs in vivo and in vitro. Biol. Reprod. 14, 354-361. Weber, K., Pringle, J.R. and Osborn, M~ (1972) Measurement of molecular weights by electrophoresis on SDS-acrylamide gel. Methods Enzymol. 26, 3-27.