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A method for chromosome preparation of guinea-pig oocytes
EnvironmentalMutagenesis
ELSEVIER
Mutation Research 334 (1995) 309-316
A method for chromosome preparation of guinea-pig oocytes P. J a c q u e t a,., L. d e S a i n t - G e o r g e s a, j. V a n k e r k o m b, L. B a u g n e t - M a h i e u a
a
Laboratory of Radiobiology, Department of Radioprotection, CEN/SCK, Boeretang 200, B-2400 Mol, Belgium b Division of Environmental Research, VITO, B-2400 Mol, Belgium Received 19 July 1994; revision received 8 November 1994; accepted 9 November 1994
Keywords: G u i n e a - p i g oocytes; C h r o m o s o m e p r e p a r a t i o n
1. Introduction
Studies performed in our laboratory aim at defining the cytogenetic effects resulting from exposure of mammalian germ cells to radiation. In a recent paper we showed that the female guinea-pig represents one of the best models for the genetic hazard of radiation in man (Jacquet et al., 1994). In contrast with other rodents studied so far, the LDs0 of guinea-pig oocytes is extremely high (about 4 Gy), like that of human oocytes. Additionally, primordial oocytes present at birth have a typical diplotene configuration, like the equivalent stage in man, while primordial oocytes from mouse or rat have a dyctiate appearance. From the genetic point of view, the primordial oocytes represent the oocyte population most at risk, since they are present throughout life and may accumulate genetic damage for considerable periods before entering maturation and being ovulated. In this respect such characteristics make the guinea-pig a more suitable model than the mouse or other rodents for studies on the genetic effects of radiation.
* Corresponding author. Tel. (32) 14-33.21.11 ext. 5184; Fax (32) 14-32.03.72.
However, performing cytogenetic studies on irradiated guinea-pig oocytes presents some difficulties. First of all, hormonal stimulation of maturation and ovulation is not easily obtained in this species, and the number of oocytes which are ovulated at each of the 17-day cycles is low (usually 2-5). The best way to obtain sufficient quantities of oocytes at metaphase I or metaphase II stages of meiosis is to culture them in vitro to the appropriate stage. Jagiello (1969) gave some information on the cytology of meiotic chromosomes in the female guinea-pig but unfortunately, she gave no detail on the techniques that she used for the isolation, culture and fixation of the oocytes. Later on, Yanagimachi (1974) described a method of culturing guinea-pig oocytes, which yielded mature oocytes capable of fertilization. However, in that paper, no attempt was made to obtain analyzable chromosome preparations of either oocyte metaphase I or II. Everybody who is familiar with the technique of Tarkowski (1966) for chromosome preparation of mouse oocytes or preimplantation embryos knows that complete metaphases with well stained and separated chromosomes are difficult to obtain, and that success partly depends on chance. One of the difficulties lies in the volume of the fixative used, which must be
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P. Jacquet et al. / Mutation Research 334 (1995) 309-316
sufficient to allow a good spreading of chromosomes, while being small enough to quickly evaporate to avoid dispersion and artefactual loss of chromosomes. In the guinea-pig, additional problems are (1) the difficulties of removing follicular cells which strongly adhere to the oocyte, (2) the necessity to remove the thick zona pellucida prior to the fixation in order to allow good spreading of the chromosomes, (3) the presence of numerous fat globules which may interfere with the quality of the preparations and (4) the high number of chromosomes (2n = 64), which makes obtaining complete metaphases still more difficult than in the mouse (2n = 40). During the last 2 years, we spent a lot of time in developing techniques to obtain good chromosome preparations from guinea-pig oocytes. For relatively large-scale investigations, such techniques had to be simplified as much as possible, while still providing reproducible results. In this respect the use of chemically defined media instead of simple biological media for the culture of the oocytes, or the use of a CO 2 incubator instead of a cheaper normal incubator, revealed no advantage. For this reason, complete details of our simplified method, now standardized, will be given in the following description.
2. Description of the method
Animals Dunkin-Hartley guinea-pigs from Bantin and Kingman Ltd (UK) are used for all our experiments. Animals are checked daily for vaginal opening for at least two cycles before use. The day the vagina is open is designated day 0 of the estrous cycle. Animals are killed on days 8-10 of the estrous cycle, when growing Graafian follicles are clearly visible at the surface of the ovaries.
Chemicals Heat-inactivated fetal calf serum is used for collecting and culturing the oocytes. Hyaluronidase from ovine testes (Fluka) and pronase, B grade (Calbiochem), are used for removing cumulus cells and the zona pellucida at the end of the culture. The enzymatic solution is
p r e p a r e d each day by dissolving 15 mg hyaluronidase and 10 mg pronase in 10 ml Dulbecco's phosphate buffered saline (PBS) without calcium, magnesium and bicarbonate (Gibco, cat nr. 042-04200). Hypotonic treatment is performed in 1% sodium citrate containing 4 m g / m l bovine serum albumin (BSA). Fixation of the oocytes is made with a freshly prepared mixture of absolute ethanol-acetic acid (3 : 1), as recommended by Tarkowski (1966).
Preparation of capillary pipettes Two types of pipettes are selected for the manipulation of the oocytes: hand-pull Pasteur capillary pipettes with a sufficiently large aperture are used for any transfer of the oocytes, while pipettes with a smaller aperture, about equivalent to the diameter of the oocyte (80-90 /zm) are useful for helping in removing the cumulus cells. Another pipette, delivering about 10/xl drops, is used for the fixation.
Culture of oocytes and fixation of chromosomes Shortly before the guinea-pigs are killed, pronase and hyaluronidase are dissolved in PBS. The tube is closed and left at room temperature until use, later at the end of the culture. The culture medium consisting of 2 microdrops of 100 /xl FCS covered with silicone or paraffin oil is prepared in Falcon dishes, one for each animal. Those culture dishes are kept at 37°C in an air atmosphere. No difference was found between this culture system and the conventional culture in an atmosphere of 5% CO 2 in air. Guinea-pigs are killed by CO 2 inhalation, and the ovaries are excised and placed in separate embryological watch glasses containing warm FCS. The ovaries are examined under incident light with a stereo-dissecting microscope. Relatively transparent areas on the ovarian surface are punctured with disposable tuberculin syringe needles to release the content of the Graafian follicles. Only healthy and large oocytes surrounded by dense layers of cumulus cells are kept for culture. They are generally dark due to the presence of many fat globules in their cytoplasm.
P. Jacquet et al. / Mutation Research 334 (1995) 309-316
Smaller and clear oocytes, surrounded only by a few layers of cumulus cells, are deleted since they are unable to resume meiosis in culture. The meiotically competent oocytes are transferred to a microdrop of FCS in the culture dishes. Most often, 20-30 oocytes per female are collected, though this number may vary from a few to more than 50. Oocytes are cultivated for 6-8 h in order to obtain M I preparations, and for 24 h if M II preparations are needed. In the guinea-pig, the disappearance of the germinal vesicle is difficult to detect because of the opacity of the cytoplasm. Examination of fixed oocytes reveals that this process and the formation of the M I spindle may occur as early as 3-5 h after the beginning of the culture. However it is very important to avoid fixation before 6 h, not only to maximize the number of oocytes that will reach the M I stage, but also to allow the migration of the spindle to the oocyte cortex. It is our experience that M I chromosomes which are fixed before the completion of this process are generally not analyzable. This probably results from the presence of the numerous fat globules that surround them in the cytoplasm and strongly interfere with their spreading. When migration of the spindle to the cortex has occurred, fat globules are preferentially accumulated at the pole opposite to that containing the M I spindle. Oocytes are picked up from the culture dish, and the tip of the pipette is carefully cleaned with absorbent paper, to remove any traces of oil. Oocytes are transferred to an embryological watch glass containing PBS with pronase and hyaluronidase, at room temperature. In the guinea-pig, cumulus cells strongly adhere to the oocyte so that treatment with hyaluronidase has to be completed by repeating pipetting of oocytes, using a pipette with a small diameter aperture. The removal of most cumulus ceils from the ooc3,tes requires some time (2-5 min). When this is achieved, examination of the oocytes is continued at the largest magnification. Digestion of the zona pellucida by pronase is a very critical step, and needs particular attention. For this reason it is advised to work at room temperature with a rather low concentration of pronase (0.1%). It is
311
our impression that higher concentrations of pronase (i.e. 0.15%) have a detrimental effect on the quality of the fixed chromosomes. The digestion of the zona pellucida is a process that begins slowly and suddenly accelerates. A space appears between oocytes and their zona pellucida, giving the impression that the oocytes diminish in size. The outlines of the oocytes become somewhat irregular and gentle pipetting of one of them will cause it to deform, confirming that the digestion of the zona pellucida is nearly achieved. Then, all oocytes are very quickly removed from the medium and carefully gathered in the center of an embryological watch glass containing the hypotonic solution. Usually the digestion of the zona pellucida will be completed in the hypotonic solution through the small quantity of pronase transferred with the oocytes. Hence, care must be taken to avoid shaking the solution which would result in rapid dilution of the enzyme. However, if needed, removal of residual cumulus cells and of the zona pellucida can be completed at the end of the hypotonic treatment through repeated pipetting. The presence in the hypotonic solution of protein (BSA) strongly reduces the weakness of the oocytes after removal of their zona pellucida, and avoids their loss during transfer on the slide. Loss of oocytes mostly happens with the use of unadapted capillary pipettes. Hypotonic treatment is prolonged for at least 15 min before the beginning of the fixation. At that time, oocytes have recovered a perfectly spherical shape, and any small differences in their size are easily visible. Oocytes which are smaller than the majority of others are discarded, since they are meiotically incompetent and would invariably show a diplotene nucleus after fixation. Fixation of the oocytes is another delicate process. A very small drop of hypotonic solution, containing a single oocyte, is placed in the center of a small square engraved on the reverse side of a grease free slide. Then, the fixative (10 /~1) is expelled on top of the oocyte. The fixative begins to spread on the slide and after a few seconds, the hypotonic microdrop containing the oocyte slightly reappears, enlarged by partial mixing with the fixative. The microdrop usually moves slowly towards one edge of the slide, while the oocyte
312
P. Jacquet et al. / Mutation Research 334 (1995) 309-316
keeps its position. This phenomenon is accelerated by expelling a second drop of fixative at the microdrop edge. This way, the aqueous microdrop is pushed to the edge of the slide by the spreading fixative. Immediately, the slide is placed vertically on a filter paper to help remove quickly the hypotonic solution and the excess fixative by absorption. A few seconds after, a third drop of fixative is quickly expelled just over the oocyte. Removal of the aqueous hypotonic solution is important for the success of the fixation since dilution of the fixative by water slows down its evaporation, facilitating artefactual loss of chromosomes during their spreading. This, together with the small volume of fixative used, considerably reduces that risk, while it also improves the quality of the fixation. Thus M I preparations where one of the 32 chromosome pairs is missing are rare and bivalents appear generally well separated, regularly cross-shaped. Loss of M II chromosomes occurs with a higher frequency than M I chromosomes. This could be due to the smaller weight of univalents compared to bivalents. Using the labelling margin of the slide as a reference for the engraved square in which the oocyte was deposited, the relative oocyte position is indicated with a pencil. All slides are stained with lacto-aceto orcein, 1-2 days after fixation.
3. Discussion
Environmental mutagens, such as radiation and a number of chemicals, may induce in the germ cells structural as well as numerical chromosome anomalies which are eventually transmitted to the progeny. Structural anomalies are usually analyzed in M I oocytes, while detection of numerical anomalies, which requires at least one meiotic division, is often performed in M II oocytes or immediately after fertilization in the one-cell embryo. For studies on the genetic hazard of radiation in man, the guinea-pig is a very useful model (Jacquet et al., 1994), and the purpose of the present work was to develop reliable techniques
for the preparation of meiotic chromosomes from this species. The method described above makes it possible to reproducibly obtain excellent chromosome preparations from M I guinea-pig oocytes (Fig. la-d). About 40% (178/460) of oocytes cultured for 6-8 h yielded analyzable M I preparations. This percentage compares favorably with the 22% analyzable M I preparations obtained by Morrison et al. (1983) for rabbit oocytes. Moreover, in our last series, and provided degenerated or remaining incompetent oocytes were discarded at the end of the hypotonic treatment, the proportion of those which yielded analyzable bivalent spreads frequently exceeded 50% and sometimes reached as high as 80-90%. These values are roughly similar to those obtained by McGaughey and Chang (1969) (55%), but somewhat lower than those reported later by Kamiguchi et al. (1976) (84.2 %), for mouse oocytes. This discrepancy partly results from the relatively high rate of guinea-pig oocytes which were already at M II after 6-8 h of culture. As evidenced by our preliminary investigations, this rate did not depend on the duration of the culture. Indeed, second meiotic divisions were found at variable frequencies, either oocytes had been cultured for different short times (7, 6, 5, 4.5, 4 or 3.5 h), or even they had been fixed directly after their collection. We found as much as 53% M II oocytes in one series fixed without preliminary culture, while 10% were found in another series cultured for 7 h (Table 1).
Table 1 Proportions of second meiotic divisions in guinea-pig oocytes fixed after various culture times Culture time (h)
Oocytes at M II/oocytes
fixed 7 6 5 4.5 4 3.5 0
2/20 1/10 11/45 3/10 3/9 0/16 9/17
Total
29/127
313
P. Jacquet et al. / M u t a t i o n Research 334 (1995) 3 0 9 - 3 1 6
4
0
t~
11,1~ ,¢.
gt ,,o,, ~
o
0
o#
b
15
i? dgP
i ii~! i i~ !iiii!iil~ iiiii~!
d Fig. 1. Guinea-pig oocytes in M I. (a) and (c) are from control females, while (b) and (d) are from females irradiated on the ovaries with 2 Gy of X-rays. Collection and culture of the oocytes occurred immediately after irradiation. (b) and (d) show a chromatid break and a chromosome translocation, respectively (arrows).
P. Jacquet et al. /Mutation Research 334 (1995) 309-316
314
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#
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.? a
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c
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P. Jacquet et al. / Mutation Research 334 (1995) 309-316
Second meiotic divisions were also reported by Edwards (1962) in oocytes from other mammalian species including man, the mouse and the baboon, fixed directly after their collection. As pointed out by the author, it is clear that most if not all of these M II oocytes had been stimulated in vivo, before removal of the ovaries. They had probably been liberated from atretic follicles. We assume that a similar phenomenon was responsible for the high proportion of M II frequently observed in preparations of guinea-pig oocytes fixed after short-term cultures. The technique described also makes it possible to obtain good chromosome preparations from M II oocytes. All oocytes selected for fixation after 24 h culture were shown to be at M II, and 36% of them (22/61) fulfilled the qualitative requirements for chromosome analysis. Most of them contained 64 univalents, representing the chromosomes both of the oocyte and of the first polar body. In the mouse oocyte, the chromosomes of the first polar body, with their fuzzy-like appearance (R6hrborn et al., 1977), differ markedly from the corkscrew-like twisted chromosomes of the oocyte. However, such a difference was rarely seen in our guinea-pig preparations. In many cases (17/22), all the chromosomes appeared mixed in a single metaphase plate and their respective origins, oocyte or polar body, could not be ascertained (Fig. 2a,b). Less often (5/22), the chromosomes were separated in sets of 32 univalents (Fig. 2c,d). The high frequency of preparations containing 64 grouped univalents should clearly constitute a handicap for studies on the induction of aneuploidy by radiation or other mutagen. The proportion of M II oocytes available for such analysis can be improved by the mechanical removal of the first polar body, just before fixation, through careful and repeated pipetting.
315
In summary, a method was developed for the in vitro maturation of guinea-pig oocytes and the preparation of their meiotic chromosomes. Some advantages of the method are that oocytes are collected and cultured in the same simple biological medium, in an air atmosphere. Culturing oocytes in an atmosphere of air represents a cheaper alternative to the conventional dioxide gas incubation. Furthermore, prior equilibration of the medium with gas, control and adjustment of its pH, are no longer required. Thus, oocytes can be cultured under less rigorous laboratory conditions. Other advantages are the excellent quality of the chromosome preparations usually obtained, and its reproducibility. This is essentially obtained by a careful control of the enzymatic treatment of oocytes at the end of the culture, by small modifications of the fixation procedure of Tarkowski (1966), and by rigorous standardization of all manipulations. These techniques will be particularly suited for studies on the induction of structural chromosome aberrations, such as translocations, in the female germ cells of the guinea-pig.
Acknowledgement This work was supported by a Research Contract from the European Communities (Contract No. FI3P-CT920005).
References Jacquet, P., J. Vankerkom and M. Lambiet-Collier (1994) The female guinea-pig, a useful model for the genetic hazard of radiation in man; preliminary results on germ cell radiosensitivity in foetal, neonatal and adult animals, Int. J. Radiat. Biol., 65, 357-367. Jagiello, G.M. (1969) Some cytologic aspects of meiosis in female guinea-pig, Chromosoma, 27, 95-101. Kamiguchi, Y., K. Funaki and K. Mikamo (1976) A new
Fig. 2. Guinea-pig oocytes in M II. (a) and (b) show the 64 chromosomes of the oocyte nucleus and of the first polar body, which are grouped. In (c) and (d), only 32 chromosomes are present, a - c are from control females, d is from a female irradiated with 2 Gy and shows two chromatid fragments (arrows).
316
P. Jacquet et al. /Mutation Research 334 (1995) 309-316
technique for chromosome study of murine oocyte, Proc. Jpn. Acad., 52, 316-319. McGaughey, R.W. and M.C. Chang (1969) Meiosis of mouse eggs before and after sperm penetration, J. Exp. Zool., 1970, 397-409. Morrison, W.D., V. Huff, L.G. Littlefield and R.J. Dufrain (1983) Chromosome preparations from rabbit preovulatory oocytes, Mutation Res., 119, 169-175. R6hrborn, G., I. Hansmann and U. Buckel (1977) Cytogenetic analysis of pre- and postovulatory oocytes and pre-implan-
tation embryos in mutagenesis of mammals, in: B.J. Kilbey, M.S. Legator, W. Nichols and C. Ramel (Eds.), Handbook of Mutagenicity Test Procedures, Elsevier, Amsterdam, pp. 301-310. Tarkowski, A.K. (1966) An air-drying method for chromosome preparations from mouse eggs, Cytogenetics, 5, 394-400. Yanagimachi, R. (1974) Maturation and fertilization in vitro of guinea-pig ovarian oocytes, J. Reprod. Fertil., 38, 485488.
ELSEVIER
Mutation Research 334 (1995) 309-316
A method for chromosome preparation of guinea-pig oocytes P. J a c q u e t a,., L. d e S a i n t - G e o r g e s a, j. V a n k e r k o m b, L. B a u g n e t - M a h i e u a
a
Laboratory of Radiobiology, Department of Radioprotection, CEN/SCK, Boeretang 200, B-2400 Mol, Belgium b Division of Environmental Research, VITO, B-2400 Mol, Belgium Received 19 July 1994; revision received 8 November 1994; accepted 9 November 1994
Keywords: G u i n e a - p i g oocytes; C h r o m o s o m e p r e p a r a t i o n
1. Introduction
Studies performed in our laboratory aim at defining the cytogenetic effects resulting from exposure of mammalian germ cells to radiation. In a recent paper we showed that the female guinea-pig represents one of the best models for the genetic hazard of radiation in man (Jacquet et al., 1994). In contrast with other rodents studied so far, the LDs0 of guinea-pig oocytes is extremely high (about 4 Gy), like that of human oocytes. Additionally, primordial oocytes present at birth have a typical diplotene configuration, like the equivalent stage in man, while primordial oocytes from mouse or rat have a dyctiate appearance. From the genetic point of view, the primordial oocytes represent the oocyte population most at risk, since they are present throughout life and may accumulate genetic damage for considerable periods before entering maturation and being ovulated. In this respect such characteristics make the guinea-pig a more suitable model than the mouse or other rodents for studies on the genetic effects of radiation.
* Corresponding author. Tel. (32) 14-33.21.11 ext. 5184; Fax (32) 14-32.03.72.
However, performing cytogenetic studies on irradiated guinea-pig oocytes presents some difficulties. First of all, hormonal stimulation of maturation and ovulation is not easily obtained in this species, and the number of oocytes which are ovulated at each of the 17-day cycles is low (usually 2-5). The best way to obtain sufficient quantities of oocytes at metaphase I or metaphase II stages of meiosis is to culture them in vitro to the appropriate stage. Jagiello (1969) gave some information on the cytology of meiotic chromosomes in the female guinea-pig but unfortunately, she gave no detail on the techniques that she used for the isolation, culture and fixation of the oocytes. Later on, Yanagimachi (1974) described a method of culturing guinea-pig oocytes, which yielded mature oocytes capable of fertilization. However, in that paper, no attempt was made to obtain analyzable chromosome preparations of either oocyte metaphase I or II. Everybody who is familiar with the technique of Tarkowski (1966) for chromosome preparation of mouse oocytes or preimplantation embryos knows that complete metaphases with well stained and separated chromosomes are difficult to obtain, and that success partly depends on chance. One of the difficulties lies in the volume of the fixative used, which must be
0165-1161/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 1 6 5 - 1 1 6 1 ( 9 4 ) 0 0 0 9 5 - 6
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P. Jacquet et al. / Mutation Research 334 (1995) 309-316
sufficient to allow a good spreading of chromosomes, while being small enough to quickly evaporate to avoid dispersion and artefactual loss of chromosomes. In the guinea-pig, additional problems are (1) the difficulties of removing follicular cells which strongly adhere to the oocyte, (2) the necessity to remove the thick zona pellucida prior to the fixation in order to allow good spreading of the chromosomes, (3) the presence of numerous fat globules which may interfere with the quality of the preparations and (4) the high number of chromosomes (2n = 64), which makes obtaining complete metaphases still more difficult than in the mouse (2n = 40). During the last 2 years, we spent a lot of time in developing techniques to obtain good chromosome preparations from guinea-pig oocytes. For relatively large-scale investigations, such techniques had to be simplified as much as possible, while still providing reproducible results. In this respect the use of chemically defined media instead of simple biological media for the culture of the oocytes, or the use of a CO 2 incubator instead of a cheaper normal incubator, revealed no advantage. For this reason, complete details of our simplified method, now standardized, will be given in the following description.
2. Description of the method
Animals Dunkin-Hartley guinea-pigs from Bantin and Kingman Ltd (UK) are used for all our experiments. Animals are checked daily for vaginal opening for at least two cycles before use. The day the vagina is open is designated day 0 of the estrous cycle. Animals are killed on days 8-10 of the estrous cycle, when growing Graafian follicles are clearly visible at the surface of the ovaries.
Chemicals Heat-inactivated fetal calf serum is used for collecting and culturing the oocytes. Hyaluronidase from ovine testes (Fluka) and pronase, B grade (Calbiochem), are used for removing cumulus cells and the zona pellucida at the end of the culture. The enzymatic solution is
p r e p a r e d each day by dissolving 15 mg hyaluronidase and 10 mg pronase in 10 ml Dulbecco's phosphate buffered saline (PBS) without calcium, magnesium and bicarbonate (Gibco, cat nr. 042-04200). Hypotonic treatment is performed in 1% sodium citrate containing 4 m g / m l bovine serum albumin (BSA). Fixation of the oocytes is made with a freshly prepared mixture of absolute ethanol-acetic acid (3 : 1), as recommended by Tarkowski (1966).
Preparation of capillary pipettes Two types of pipettes are selected for the manipulation of the oocytes: hand-pull Pasteur capillary pipettes with a sufficiently large aperture are used for any transfer of the oocytes, while pipettes with a smaller aperture, about equivalent to the diameter of the oocyte (80-90 /zm) are useful for helping in removing the cumulus cells. Another pipette, delivering about 10/xl drops, is used for the fixation.
Culture of oocytes and fixation of chromosomes Shortly before the guinea-pigs are killed, pronase and hyaluronidase are dissolved in PBS. The tube is closed and left at room temperature until use, later at the end of the culture. The culture medium consisting of 2 microdrops of 100 /xl FCS covered with silicone or paraffin oil is prepared in Falcon dishes, one for each animal. Those culture dishes are kept at 37°C in an air atmosphere. No difference was found between this culture system and the conventional culture in an atmosphere of 5% CO 2 in air. Guinea-pigs are killed by CO 2 inhalation, and the ovaries are excised and placed in separate embryological watch glasses containing warm FCS. The ovaries are examined under incident light with a stereo-dissecting microscope. Relatively transparent areas on the ovarian surface are punctured with disposable tuberculin syringe needles to release the content of the Graafian follicles. Only healthy and large oocytes surrounded by dense layers of cumulus cells are kept for culture. They are generally dark due to the presence of many fat globules in their cytoplasm.
P. Jacquet et al. / Mutation Research 334 (1995) 309-316
Smaller and clear oocytes, surrounded only by a few layers of cumulus cells, are deleted since they are unable to resume meiosis in culture. The meiotically competent oocytes are transferred to a microdrop of FCS in the culture dishes. Most often, 20-30 oocytes per female are collected, though this number may vary from a few to more than 50. Oocytes are cultivated for 6-8 h in order to obtain M I preparations, and for 24 h if M II preparations are needed. In the guinea-pig, the disappearance of the germinal vesicle is difficult to detect because of the opacity of the cytoplasm. Examination of fixed oocytes reveals that this process and the formation of the M I spindle may occur as early as 3-5 h after the beginning of the culture. However it is very important to avoid fixation before 6 h, not only to maximize the number of oocytes that will reach the M I stage, but also to allow the migration of the spindle to the oocyte cortex. It is our experience that M I chromosomes which are fixed before the completion of this process are generally not analyzable. This probably results from the presence of the numerous fat globules that surround them in the cytoplasm and strongly interfere with their spreading. When migration of the spindle to the cortex has occurred, fat globules are preferentially accumulated at the pole opposite to that containing the M I spindle. Oocytes are picked up from the culture dish, and the tip of the pipette is carefully cleaned with absorbent paper, to remove any traces of oil. Oocytes are transferred to an embryological watch glass containing PBS with pronase and hyaluronidase, at room temperature. In the guinea-pig, cumulus cells strongly adhere to the oocyte so that treatment with hyaluronidase has to be completed by repeating pipetting of oocytes, using a pipette with a small diameter aperture. The removal of most cumulus ceils from the ooc3,tes requires some time (2-5 min). When this is achieved, examination of the oocytes is continued at the largest magnification. Digestion of the zona pellucida by pronase is a very critical step, and needs particular attention. For this reason it is advised to work at room temperature with a rather low concentration of pronase (0.1%). It is
311
our impression that higher concentrations of pronase (i.e. 0.15%) have a detrimental effect on the quality of the fixed chromosomes. The digestion of the zona pellucida is a process that begins slowly and suddenly accelerates. A space appears between oocytes and their zona pellucida, giving the impression that the oocytes diminish in size. The outlines of the oocytes become somewhat irregular and gentle pipetting of one of them will cause it to deform, confirming that the digestion of the zona pellucida is nearly achieved. Then, all oocytes are very quickly removed from the medium and carefully gathered in the center of an embryological watch glass containing the hypotonic solution. Usually the digestion of the zona pellucida will be completed in the hypotonic solution through the small quantity of pronase transferred with the oocytes. Hence, care must be taken to avoid shaking the solution which would result in rapid dilution of the enzyme. However, if needed, removal of residual cumulus cells and of the zona pellucida can be completed at the end of the hypotonic treatment through repeated pipetting. The presence in the hypotonic solution of protein (BSA) strongly reduces the weakness of the oocytes after removal of their zona pellucida, and avoids their loss during transfer on the slide. Loss of oocytes mostly happens with the use of unadapted capillary pipettes. Hypotonic treatment is prolonged for at least 15 min before the beginning of the fixation. At that time, oocytes have recovered a perfectly spherical shape, and any small differences in their size are easily visible. Oocytes which are smaller than the majority of others are discarded, since they are meiotically incompetent and would invariably show a diplotene nucleus after fixation. Fixation of the oocytes is another delicate process. A very small drop of hypotonic solution, containing a single oocyte, is placed in the center of a small square engraved on the reverse side of a grease free slide. Then, the fixative (10 /~1) is expelled on top of the oocyte. The fixative begins to spread on the slide and after a few seconds, the hypotonic microdrop containing the oocyte slightly reappears, enlarged by partial mixing with the fixative. The microdrop usually moves slowly towards one edge of the slide, while the oocyte
312
P. Jacquet et al. / Mutation Research 334 (1995) 309-316
keeps its position. This phenomenon is accelerated by expelling a second drop of fixative at the microdrop edge. This way, the aqueous microdrop is pushed to the edge of the slide by the spreading fixative. Immediately, the slide is placed vertically on a filter paper to help remove quickly the hypotonic solution and the excess fixative by absorption. A few seconds after, a third drop of fixative is quickly expelled just over the oocyte. Removal of the aqueous hypotonic solution is important for the success of the fixation since dilution of the fixative by water slows down its evaporation, facilitating artefactual loss of chromosomes during their spreading. This, together with the small volume of fixative used, considerably reduces that risk, while it also improves the quality of the fixation. Thus M I preparations where one of the 32 chromosome pairs is missing are rare and bivalents appear generally well separated, regularly cross-shaped. Loss of M II chromosomes occurs with a higher frequency than M I chromosomes. This could be due to the smaller weight of univalents compared to bivalents. Using the labelling margin of the slide as a reference for the engraved square in which the oocyte was deposited, the relative oocyte position is indicated with a pencil. All slides are stained with lacto-aceto orcein, 1-2 days after fixation.
3. Discussion
Environmental mutagens, such as radiation and a number of chemicals, may induce in the germ cells structural as well as numerical chromosome anomalies which are eventually transmitted to the progeny. Structural anomalies are usually analyzed in M I oocytes, while detection of numerical anomalies, which requires at least one meiotic division, is often performed in M II oocytes or immediately after fertilization in the one-cell embryo. For studies on the genetic hazard of radiation in man, the guinea-pig is a very useful model (Jacquet et al., 1994), and the purpose of the present work was to develop reliable techniques
for the preparation of meiotic chromosomes from this species. The method described above makes it possible to reproducibly obtain excellent chromosome preparations from M I guinea-pig oocytes (Fig. la-d). About 40% (178/460) of oocytes cultured for 6-8 h yielded analyzable M I preparations. This percentage compares favorably with the 22% analyzable M I preparations obtained by Morrison et al. (1983) for rabbit oocytes. Moreover, in our last series, and provided degenerated or remaining incompetent oocytes were discarded at the end of the hypotonic treatment, the proportion of those which yielded analyzable bivalent spreads frequently exceeded 50% and sometimes reached as high as 80-90%. These values are roughly similar to those obtained by McGaughey and Chang (1969) (55%), but somewhat lower than those reported later by Kamiguchi et al. (1976) (84.2 %), for mouse oocytes. This discrepancy partly results from the relatively high rate of guinea-pig oocytes which were already at M II after 6-8 h of culture. As evidenced by our preliminary investigations, this rate did not depend on the duration of the culture. Indeed, second meiotic divisions were found at variable frequencies, either oocytes had been cultured for different short times (7, 6, 5, 4.5, 4 or 3.5 h), or even they had been fixed directly after their collection. We found as much as 53% M II oocytes in one series fixed without preliminary culture, while 10% were found in another series cultured for 7 h (Table 1).
Table 1 Proportions of second meiotic divisions in guinea-pig oocytes fixed after various culture times Culture time (h)
Oocytes at M II/oocytes
fixed 7 6 5 4.5 4 3.5 0
2/20 1/10 11/45 3/10 3/9 0/16 9/17
Total
29/127
313
P. Jacquet et al. / M u t a t i o n Research 334 (1995) 3 0 9 - 3 1 6
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d Fig. 1. Guinea-pig oocytes in M I. (a) and (c) are from control females, while (b) and (d) are from females irradiated on the ovaries with 2 Gy of X-rays. Collection and culture of the oocytes occurred immediately after irradiation. (b) and (d) show a chromatid break and a chromosome translocation, respectively (arrows).
P. Jacquet et al. /Mutation Research 334 (1995) 309-316
314
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P. Jacquet et al. / Mutation Research 334 (1995) 309-316
Second meiotic divisions were also reported by Edwards (1962) in oocytes from other mammalian species including man, the mouse and the baboon, fixed directly after their collection. As pointed out by the author, it is clear that most if not all of these M II oocytes had been stimulated in vivo, before removal of the ovaries. They had probably been liberated from atretic follicles. We assume that a similar phenomenon was responsible for the high proportion of M II frequently observed in preparations of guinea-pig oocytes fixed after short-term cultures. The technique described also makes it possible to obtain good chromosome preparations from M II oocytes. All oocytes selected for fixation after 24 h culture were shown to be at M II, and 36% of them (22/61) fulfilled the qualitative requirements for chromosome analysis. Most of them contained 64 univalents, representing the chromosomes both of the oocyte and of the first polar body. In the mouse oocyte, the chromosomes of the first polar body, with their fuzzy-like appearance (R6hrborn et al., 1977), differ markedly from the corkscrew-like twisted chromosomes of the oocyte. However, such a difference was rarely seen in our guinea-pig preparations. In many cases (17/22), all the chromosomes appeared mixed in a single metaphase plate and their respective origins, oocyte or polar body, could not be ascertained (Fig. 2a,b). Less often (5/22), the chromosomes were separated in sets of 32 univalents (Fig. 2c,d). The high frequency of preparations containing 64 grouped univalents should clearly constitute a handicap for studies on the induction of aneuploidy by radiation or other mutagen. The proportion of M II oocytes available for such analysis can be improved by the mechanical removal of the first polar body, just before fixation, through careful and repeated pipetting.
315
In summary, a method was developed for the in vitro maturation of guinea-pig oocytes and the preparation of their meiotic chromosomes. Some advantages of the method are that oocytes are collected and cultured in the same simple biological medium, in an air atmosphere. Culturing oocytes in an atmosphere of air represents a cheaper alternative to the conventional dioxide gas incubation. Furthermore, prior equilibration of the medium with gas, control and adjustment of its pH, are no longer required. Thus, oocytes can be cultured under less rigorous laboratory conditions. Other advantages are the excellent quality of the chromosome preparations usually obtained, and its reproducibility. This is essentially obtained by a careful control of the enzymatic treatment of oocytes at the end of the culture, by small modifications of the fixation procedure of Tarkowski (1966), and by rigorous standardization of all manipulations. These techniques will be particularly suited for studies on the induction of structural chromosome aberrations, such as translocations, in the female germ cells of the guinea-pig.
Acknowledgement This work was supported by a Research Contract from the European Communities (Contract No. FI3P-CT920005).
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Fig. 2. Guinea-pig oocytes in M II. (a) and (b) show the 64 chromosomes of the oocyte nucleus and of the first polar body, which are grouped. In (c) and (d), only 32 chromosomes are present, a - c are from control females, d is from a female irradiated with 2 Gy and shows two chromatid fragments (arrows).
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technique for chromosome study of murine oocyte, Proc. Jpn. Acad., 52, 316-319. McGaughey, R.W. and M.C. Chang (1969) Meiosis of mouse eggs before and after sperm penetration, J. Exp. Zool., 1970, 397-409. Morrison, W.D., V. Huff, L.G. Littlefield and R.J. Dufrain (1983) Chromosome preparations from rabbit preovulatory oocytes, Mutation Res., 119, 169-175. R6hrborn, G., I. Hansmann and U. Buckel (1977) Cytogenetic analysis of pre- and postovulatory oocytes and pre-implan-
tation embryos in mutagenesis of mammals, in: B.J. Kilbey, M.S. Legator, W. Nichols and C. Ramel (Eds.), Handbook of Mutagenicity Test Procedures, Elsevier, Amsterdam, pp. 301-310. Tarkowski, A.K. (1966) An air-drying method for chromosome preparations from mouse eggs, Cytogenetics, 5, 394-400. Yanagimachi, R. (1974) Maturation and fertilization in vitro of guinea-pig ovarian oocytes, J. Reprod. Fertil., 38, 485488.