- Email: [email protected]
[5] Isolation and culture of oocytes
[5]
OOCYTE
ISOLATION
AND CULTURE
77
introduced into a 10-/~1 microcapillary tube which has been flame-sealed at one end and cut down to a suitable length. 3. Centrifuge the sample at 10,000 rpm for 10 min at 18-20 °. 4. Remove the pellet from the tube. This is most easily done by first submerging the entire tube in a dish of culture medium, and then introducing a fine pipette between the pellet and the inside of the tube. The pellet can then be dislodged with gentle currents of medium and washed out into the dish. 5. The pellet should be allowed to recover in an incubator for several hours without further handling. During this time the cells begin to attach to each other, and the pellet assumes some degree of structural integrity which enables it to be handled without breaking. 6. After the recovery period, the pellet can be cultured according to any protocol suitable for embryonic gonads.
Phytohemagglutininfor Cell Aggregations. Make up a stock solution of phytohemagglutinin in distilled water, as directed by the suppliers, and dilute this 1 : 100 in a suitable medium.
[5] I s o l a t i o n a n d C u l t u r e o f O o c y t e s
By JOHN J. EPPIG and EVELYN E. TELFER Introduction Oocyte growth occurs primarily in preantral follicles when the oocyte is arrested in prophase I of meiosis and is incompetent of resuming meiosis. About the time that a follicular antrum is formed, the oocyte nears completion of its growth phase and becomes competent of resuming meiosis. Therefore, antral follicles are the source of fully grown, immature, germinal vesicle (GV)-stage oocytes capable of undergoing spontaneous gonadotropin-independent maturation, 1 and preantral follicles are the source of growing, immature GV-stage oocytes incapable of spontaneous maturation i Maturation is defined here as a process encompassing both the nuclear and the cytoplasmic events that occur in oocytes to prepare them for fertilization. The first morphological indication of maturation is the dissolution or breakdown of the nuclear envelope (germinal vesicle breakdown, GVB). Meiosis then proceeds to metaphase II where it is once again arrested, this time until sperm penetration initiates the completion of meiosis. Cytoplasmic events also occur that promote pronuclear formation and preparation for early embryogenesis. In this chapter, the terms oocyte or immature oocyte refer to GV-stage, primary oocytes, and the terms mature oocyte, ovum, or egg refer to secondary oocytes ready for fertilization.
METHODS IN ENZYMOLOGY, VOL. 225
Copyright © 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.
78
GERM CELLS AND EMBRYOS
[5]
without further development. This chapter describes techniques for the collection and culture of both meiosis-incompetent and meiosis-competent oocytes to produce eggs able to undergo fertilization and development to live young. Isolation and Maturation of Fully Grown, Meiotically Competent Germinal Vesicle-Stage Oocytes
Animals Follicular development is stimulated in immature mice, around 24 days old, by an intraperitoneal injection of 5 IU of pregnant mare serum gonadotropin (PMSG) 48 hr before sacrificing them by cervical dislocation. Adult cycling mice can also be used, but the yield of oocytes is much smaller.
Isolation and Collection of Oocyte-Cumulus Cell Complexes Ovaries are removed from animals and immersed in Waymouth MB752/1 medium (Table I) supplemented with 0.23 mM pyruvic acid, 50 rag/liter streptomycin sulfate, 75 mg/liter penicillin G (potassium salt), and 5% (v/v) fetal bovine serum (FBS). Initial studies of the developmental capacity of oocytes matured in vitro utilized minimum essential medium
TABLE I PREPARATION OF WAYMOUTH MEDIUM MB752/1 FOR OOCYTE GROWTH AND MATURATIONa Component MB752/1 powder b NaHCO3 Pencillin G, potassium salt Streptomycin sulfate Pyruvic acid, sodium salt Fetal bovine serum
Amount Contents of 2240 75 50 25 50
1 liter kit mg mg mg mg ml
Dissolve MB752/1 powder kit in about 750 ml of water, then dissolve the NaHCO3. Add the remaining components, except for the serum, and bring to 1 liter volume. Bubble 5-5-90 gas through the medium for 5 to 10 min, then add the serum and sterilize by filtration. Perfuse the storage bottle with 5-5-90 gas before refrigeration. Do not store longer than 2 weeks. b GIBCO, 430 1400EB.
[5]
OOCYTE ISOLATION AND CULTURE
79
FIG. 1. Photomicrograph of oocyte-cumulus cell complexes (solid arrows) isolated from the antral folliclesof a 24-day-oldmouse. These can be confused with large preantral follicles (empty arrow) that are sometimes released from the ovary while puncturing the antral follicles with a needle. Note that the periphery of the preantral follicle is much smoother than that of the oocyte-cumulus cell complex. The cumulus cell-enclosed oocytes from antral follicles will undergo spontaneous maturation, but the growing oocyte in the preantral follicle will not. Bar: 100/~m.
(MEM), 2 but subsequent studies, 3 in which several media were tested, showed that oocytes matured in W a y m o u t h MB752/1 medium had the highest percentage of subsequent development to the blastocyst stage. The large antral follicles are punctured using 25-gauge needles mounted on 1-ml syringe barrels. Wipe the needles with a Kimwipe saturated with 75% ethanol before use to remove lubricant. Do not use plastic tissue culture dishes, because they have been treated to encourage cell adhesion and adhesion makes manipulations difficult; Falcon petri dishes (No. 1008) are recommended. Collect the o o c y t e - c u m u l u s cell complexes using glass pipettes drawn from 4 mm Pyrex glass tubing. Select only complexes having GV-stage oocytes completely enclosed by cumulus cells. Be careful to avoid selecting preantral follicles that are sometimes released from the ovary during antral follicle puncture (Fig. 1). Wash the complexes by serially transferring them through four dishes containing 2.5 ml of maturation medium.
Oocyte Maturation Place the washed complexes in a petri dish containing 2.5 ml of maturation medium that is also supplemented with 1 /~g/ml biological grade follicle-stimulating hormone (FSH). Place the dish in a modular incubator (Billups-Rothenberg) that can be flushed with a gas mixture of 5% 02, 5% CO2, and 90% N 2 (v/v; hereafter referred to as 5-5-90 gas). Incubate 2 A. C. Schroeder and J. J. Eppig, Dev. Biol. 102, 493 (1984). 3 j. j. M. Van de Sandt, A. C. Schroeder, and J. J. Eppig, Mol. Reprod. Dev. 25, 164 (1990).
80
GERM CELLS AND EMBRYOS
[5]
at 37 ° for approximately 17 hr. Make sure the complexes are not clumped in the maturation dish, as aggregation has a deleterious effect on oocyte maturation. After maturation, wash the complexes in the fertilization medium and inseminate immediately. The medium used for the collection and maturation ofoocytes is usually supplemented with 5% FBS to prevent hardening of the zona pellucida, 4-6 an event that would render the zona pellucida resistant to sperm penetration. Ifa serum-free medium is needed, medium supplemented with 1 mg/ml fetuin can be used. 7 Stock solutions of fetuin (10 mg/ml; Spiro preparation, GIBCO, Grand Island, NY) in maturation medium should be dialyzed 3 times against maturation medium (10 times the volume of the dialyzate) before use. Stock solutions of fetuin can be stored for 2 weeks at 4 °. Cumulus cell-enclosed GV-stage oocytes can be manipulated, by microinjection, for example, before maturation begins. Add 250/xM dibutyryl cyclic adenosine monophosphate or 100/~M 3-isobutyl-l-methylxanthine to the maturation medium to maintain meiotic arrest while manipulating the oocytes, then wash the complexes in maturation medium and proceed with oocyte maturation as described above. 8-~° E x p e c t e d Results
The embryonic developmental competence of mouse oocytes matured in oitro will vary according to the age of the donor mice,ll the hormonal
status of the mice donating the GV-stage oocytes, ~2'~3 FSH treatment of the maturing oocytes in vitro, ~1 and the presence or absence of serum in the oocyte maturation medium.7'14 The effect of the genotype of the oocyte donor has not been assessed. Nevertheless, when PMSG-primed (C57BL/ 6J × SJL/J)F 1 mice 24 to 26 days old provide the GV-stage oocytes for maturation in medium supplemented with FBS, the frequency of fertilization and cleavage to the 2-cell stage should be 75 to 95%. Moreover, 65 to 4 M. DeFelici, A. Salustri, and G. Siracusa, Gamete Res. 12, 227 (1985). 5 j. G. Gianfortoni and B. J. Gulyas, Gamete Res. 11, 59 (1985). 6 S. M. Downs, A. C. Schroeder, and J. J. Eppig, Gamete Res. 15, 115 (1986). 7 A. C. Schroeder, R. M. Schultz, G. S. Kopf, F. R. Taylor, R. B. Becker, and J. J. Eppig, Biol. Reprod. 43, 891 (1990). 8 W. K. Cho, S. Stern, and J. D. Biggers, J. Exp. Zool. 187, 383 (1974). 9 C. Magnusson and T. Hillensjo, J. Exp. Zool. 201, 138 (1977). 10 S. M. Downs, A. C. Schroeder, and J. J. Eppig, Gamete Res. 15, 305 (1986). u j. j. Eppig and A. C. Schroeder, Biol. Reprod. 41, 268 (1989). 12 j. j. Eppig, A. C. Schroeder, and M. J. O'Brien, J. Reprod. Fertil. 95, 119 (1992). 13 A. C. Schroeder and J. J. Eppig, Gamete Res. 24, 81 (1989). 14j. j. Eppig, K. Wigglesworth, and M. J. O'Brien, Mol. Reprod. Dev. 32, 33 (1992).
[5]
OOCYTE ISOLATION AND CULTURE
81
85% of the 2-cell stage embryos should develop to the expanded blastocyst stage. Between 30 and 45% of the 2-cell stage embryos transferred to the oviducts of foster mothers should develop to live offspring.
Isolation, Growth, and Maturation of Growing, Meiotically Incompetent Germinal Vesicle-Stage Oocytes
Animals Large numbers of oocyte-granulosa cell complexes can be isolated from the preantral follicles of 12-day-old mice. Use of the immature mice provides ovaries that are readily dissociated with collagenase and also relatively homogeneous populations of preantral follicles.
Isolation and Collection of Oocyte-Granulosa Cell Complexes of Preantral Follicles Ovaries from 5 to 15 mice are immersed in 3.5 ml of culture medium containing 1 to 2 mg/ml crude collagenase (Worthington type CLS-1, Freehold, N J, for example) and 0.02 mg/ml DNase (sterilized by filtration) and then are placed on a slide warming tray maintained at 35° and covered with a plexiglass box that is constantly flushed with 5-5-90 gas. After about 10 min, the ovaries are vigorously drawn in and out of an Eppendorf micropipette set at 1.0 ml and equipped with a sterile blue tip until the ovaries begin to dissociate. The ovaries are returned to the warming tray for 10-min intervals, at which times the dissociating ovaries are repeatedly drawn in and out of a micropipette set at 0.1 ml and equipped with a sterile yellow tip. This process is repeated until the ovaries are completely dissociated into individual oocyte-granulosa cell complexes. This is usually completed within 40 min. The complexes are washed by swirling them together in the center of the petri dish, drawing off the cloud of debris that overhangs the settling complexes, transferring the complexes to another petri dish containing 3.0 ml of medium, and repeating this process until the preparation is free of ovarian debris, usually four or five transfers. This procedure results in a preparation such as that shown in Fig. 2. The complexes each consist of an oocyte, which is in mid-growth phase and incompetent of undergoing GV breakdown without further development, surrounded by one to three layers of granulosa cells. Most of the primitive theca layers and the basal lamina are removed by the collagenase digestion.
82
GERM CELLS AND EMBRYOS
[5]
FI6. 2. Photomicrograph of preantral follicles produced by dissociation of the ovaries of 12-day-old mice using collagenase/DNase. Bar: 100/~m.
Oocyte Growth The oocyte-granulosa cell complexes are distributed to the surface of either Transwell-COL membranes (Costar, Cambridge, MA; 3.0/zm pore size) or a rat tail collagen matrix prepared in the following way. 15'16
Preparation of Rat Tail Collagen 1. Tails obtained from rats can be frozen until needed for preparation of collagen. They are sterilized overnight in 70% (v/v) ethanol immediately before use. 2. Dissect out tendons using two pairs of artery forceps. Starting from the tip of the tail clamp one end with artery forceps and clamp approximately 3 cm away from the other pair. After bending and pulling both pairs of forceps, the tendons will come loose from the skin. Cut the tendons loose and place them in a small beaker with 70% ethanol. 3. Wash tendons in sterile distilled water and blot dry with sterile filter paper. 15 R. I. Ehrmann and G. O. Gey, J. Natl. Cancer Inst. 16, 1375 (1956). 16 M. Chambard, J. Gabrion, and J. Mauchamp, J. Cell Biol. 91, 157 (1981).
[5]
OOCYTE ISOLATION AND CULTURE
83
4. Weigh tendons and place them in a large bottle with a sterile magnetic stir bar. 5. Add 100 ml of 1 : 1000 acetic acid-water per gram of tendons. 6. Stir tendons on a magnetic stirring plate in a cold room for 48 hr. 7. Transfer collagen to 50-ml centrifuge tubes and centrifuge at 4000 rpm at 4° for 60 min. 8. Dialyze the collagen solution against a 10-fold volume of 1 : 1000 acetic acid-water 3 times for 24 hr each time. 9. Transfer collagen to new 50-ml centrifuge tubes on ice. 10. Store collagen in a refrigerator or freezer.
Preparation of Collagen Gel Matrix for Culture 1. Keep all components on ice. Mix the following in order: 1.6 ml of rat tail collagen, 0.2 ml Dulbecco's phosphate-buffered saline (PBS), and 0.2 ml of 10 × culture medium. 2. Add 70 to 100/zl of an ice-cold 0.5 M NaOH solution. Each lot of collagen is a little different, so the exact amount should be determined. The mixture should be homogeneous and pink in color. 3. Using a micropipette equipped with a sterile blue tip, add 0.250 ml of the collagen solution to the bottom of a well in a 24-well tissue culture cluster dish and distribute evenly. Keep cold during this process. 4. Set the culture dish on the slide warmer and allow the collagen to gel (>15 min). 5. Overlay the collagen gel matrix with about 1 ml of PBS and change every 5 min for 30 min. Add and remove the solutions very gently. 6. Overlay the collagen gel matrix with culture medium and incubate on the warming tray for 5 to 10 min. 7. Discard the medium and replace with 1 ml of fresh culture medium and distribute the oocyte-granulosa cell complexes. Cultures are incubated at 37° in modular incubation chambers flushed with 5-5-90 gas and fed by exchanging approximately one-half of the medium with fresh medium every second day.
Maturation of in Vitro Grown Oocytes After culturing the oocyte-granulosa cell complexes for 10 to 14 days, remove them from the surface of the Transwell-COL membrane or collagen gel matrix. This is done by sharply snapping the side of the dish with a fingernail to jolt the complexes free of the substratum. The complexes are collected with a drawn glass micropipette and matured as described above. After maturation, wash the complexes in the medium used for fertilization and inseminate immediately.
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[6]
Expected Results When oocyte-granulosa cell complexes are isolated from the preantral follicles of 12-day-old (C57BL/6J × SJL/J)F 1 mice and cultured for 10 days in FBS-supplemented medium, it should be anticipated that 60 to 80% of the oocytes will acquire competence to mature. Of the mature eggs, 50 to 60% will cleave to the 2-cell stage after insemination, and 55 to 70% of the 2-cell stage embryos will develop to the expanded blastocyst stage. Between 10 and 15% of the 2-cell stage embryos transferred to the oviducts of foster mothers should develop to live offspring. The oocyte-granulosa cell complexes of preantral follicles can also be grown in serum-free medium supplemented with 1 mg/ml fetuin, 5/zg/ml insulin, 5 /zg/ml transferrin, and 5 ng/ml selenium (ITS; Collaborative Research, Bedford, MA). Under these conditions, fewer oocytes competent of subsequent development to the blastocyst stage are produced.14 These results suggest that serum-born growth factors are important for optimal oocyte development.
[6] Purification, C u l t u r e , a n d F r a c t i o n a t i o n of S p e r m a t o g e n i c Cells
By ANTHONYR. BELLVI~ Introduction The adult mammalian testis contains multiple populations of somatic and germinal cells that differ markedly in their stages of growth and differentiation. Germ cells, the major cell type of the testis, undergo a complex and fascinating process of differentiation. 1The process, spermatogenesis, starts with the renewal of the rare spermatogonial stem cells (A0/As), followed by the proliferative sequence of types Apr, Aal, AI_ 4 , intermediate, and type B spermatogonia (Fig. 1). The latter form primary spermatocytes, which enter a prolonged meiotic prophase to effect pairing of homologous chromosomes, genetic recombination, and, with the two reduction divisions, random segregation of chromosomes to yield haploid spermatids. Then, during spermiogenesis, the spermatids undergo a remarkable sequence of differentiation that includes condensing the chromatin, reorganizing and shaping the nucleus, forming an acrosome replete with enzymes to aid fertilization, and assembling a tail with its microtubule I A. R. Bellv6,
Oxford Reo. Reprod. Biol. 1, 159 (1979).
METHODS IN ENZYMOLOGY, VOL. 225
Copyright © 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.
OOCYTE
ISOLATION
AND CULTURE
77
introduced into a 10-/~1 microcapillary tube which has been flame-sealed at one end and cut down to a suitable length. 3. Centrifuge the sample at 10,000 rpm for 10 min at 18-20 °. 4. Remove the pellet from the tube. This is most easily done by first submerging the entire tube in a dish of culture medium, and then introducing a fine pipette between the pellet and the inside of the tube. The pellet can then be dislodged with gentle currents of medium and washed out into the dish. 5. The pellet should be allowed to recover in an incubator for several hours without further handling. During this time the cells begin to attach to each other, and the pellet assumes some degree of structural integrity which enables it to be handled without breaking. 6. After the recovery period, the pellet can be cultured according to any protocol suitable for embryonic gonads.
Phytohemagglutininfor Cell Aggregations. Make up a stock solution of phytohemagglutinin in distilled water, as directed by the suppliers, and dilute this 1 : 100 in a suitable medium.
[5] I s o l a t i o n a n d C u l t u r e o f O o c y t e s
By JOHN J. EPPIG and EVELYN E. TELFER Introduction Oocyte growth occurs primarily in preantral follicles when the oocyte is arrested in prophase I of meiosis and is incompetent of resuming meiosis. About the time that a follicular antrum is formed, the oocyte nears completion of its growth phase and becomes competent of resuming meiosis. Therefore, antral follicles are the source of fully grown, immature, germinal vesicle (GV)-stage oocytes capable of undergoing spontaneous gonadotropin-independent maturation, 1 and preantral follicles are the source of growing, immature GV-stage oocytes incapable of spontaneous maturation i Maturation is defined here as a process encompassing both the nuclear and the cytoplasmic events that occur in oocytes to prepare them for fertilization. The first morphological indication of maturation is the dissolution or breakdown of the nuclear envelope (germinal vesicle breakdown, GVB). Meiosis then proceeds to metaphase II where it is once again arrested, this time until sperm penetration initiates the completion of meiosis. Cytoplasmic events also occur that promote pronuclear formation and preparation for early embryogenesis. In this chapter, the terms oocyte or immature oocyte refer to GV-stage, primary oocytes, and the terms mature oocyte, ovum, or egg refer to secondary oocytes ready for fertilization.
METHODS IN ENZYMOLOGY, VOL. 225
Copyright © 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.
78
GERM CELLS AND EMBRYOS
[5]
without further development. This chapter describes techniques for the collection and culture of both meiosis-incompetent and meiosis-competent oocytes to produce eggs able to undergo fertilization and development to live young. Isolation and Maturation of Fully Grown, Meiotically Competent Germinal Vesicle-Stage Oocytes
Animals Follicular development is stimulated in immature mice, around 24 days old, by an intraperitoneal injection of 5 IU of pregnant mare serum gonadotropin (PMSG) 48 hr before sacrificing them by cervical dislocation. Adult cycling mice can also be used, but the yield of oocytes is much smaller.
Isolation and Collection of Oocyte-Cumulus Cell Complexes Ovaries are removed from animals and immersed in Waymouth MB752/1 medium (Table I) supplemented with 0.23 mM pyruvic acid, 50 rag/liter streptomycin sulfate, 75 mg/liter penicillin G (potassium salt), and 5% (v/v) fetal bovine serum (FBS). Initial studies of the developmental capacity of oocytes matured in vitro utilized minimum essential medium
TABLE I PREPARATION OF WAYMOUTH MEDIUM MB752/1 FOR OOCYTE GROWTH AND MATURATIONa Component MB752/1 powder b NaHCO3 Pencillin G, potassium salt Streptomycin sulfate Pyruvic acid, sodium salt Fetal bovine serum
Amount Contents of 2240 75 50 25 50
1 liter kit mg mg mg mg ml
Dissolve MB752/1 powder kit in about 750 ml of water, then dissolve the NaHCO3. Add the remaining components, except for the serum, and bring to 1 liter volume. Bubble 5-5-90 gas through the medium for 5 to 10 min, then add the serum and sterilize by filtration. Perfuse the storage bottle with 5-5-90 gas before refrigeration. Do not store longer than 2 weeks. b GIBCO, 430 1400EB.
[5]
OOCYTE ISOLATION AND CULTURE
79
FIG. 1. Photomicrograph of oocyte-cumulus cell complexes (solid arrows) isolated from the antral folliclesof a 24-day-oldmouse. These can be confused with large preantral follicles (empty arrow) that are sometimes released from the ovary while puncturing the antral follicles with a needle. Note that the periphery of the preantral follicle is much smoother than that of the oocyte-cumulus cell complex. The cumulus cell-enclosed oocytes from antral follicles will undergo spontaneous maturation, but the growing oocyte in the preantral follicle will not. Bar: 100/~m.
(MEM), 2 but subsequent studies, 3 in which several media were tested, showed that oocytes matured in W a y m o u t h MB752/1 medium had the highest percentage of subsequent development to the blastocyst stage. The large antral follicles are punctured using 25-gauge needles mounted on 1-ml syringe barrels. Wipe the needles with a Kimwipe saturated with 75% ethanol before use to remove lubricant. Do not use plastic tissue culture dishes, because they have been treated to encourage cell adhesion and adhesion makes manipulations difficult; Falcon petri dishes (No. 1008) are recommended. Collect the o o c y t e - c u m u l u s cell complexes using glass pipettes drawn from 4 mm Pyrex glass tubing. Select only complexes having GV-stage oocytes completely enclosed by cumulus cells. Be careful to avoid selecting preantral follicles that are sometimes released from the ovary during antral follicle puncture (Fig. 1). Wash the complexes by serially transferring them through four dishes containing 2.5 ml of maturation medium.
Oocyte Maturation Place the washed complexes in a petri dish containing 2.5 ml of maturation medium that is also supplemented with 1 /~g/ml biological grade follicle-stimulating hormone (FSH). Place the dish in a modular incubator (Billups-Rothenberg) that can be flushed with a gas mixture of 5% 02, 5% CO2, and 90% N 2 (v/v; hereafter referred to as 5-5-90 gas). Incubate 2 A. C. Schroeder and J. J. Eppig, Dev. Biol. 102, 493 (1984). 3 j. j. M. Van de Sandt, A. C. Schroeder, and J. J. Eppig, Mol. Reprod. Dev. 25, 164 (1990).
80
GERM CELLS AND EMBRYOS
[5]
at 37 ° for approximately 17 hr. Make sure the complexes are not clumped in the maturation dish, as aggregation has a deleterious effect on oocyte maturation. After maturation, wash the complexes in the fertilization medium and inseminate immediately. The medium used for the collection and maturation ofoocytes is usually supplemented with 5% FBS to prevent hardening of the zona pellucida, 4-6 an event that would render the zona pellucida resistant to sperm penetration. Ifa serum-free medium is needed, medium supplemented with 1 mg/ml fetuin can be used. 7 Stock solutions of fetuin (10 mg/ml; Spiro preparation, GIBCO, Grand Island, NY) in maturation medium should be dialyzed 3 times against maturation medium (10 times the volume of the dialyzate) before use. Stock solutions of fetuin can be stored for 2 weeks at 4 °. Cumulus cell-enclosed GV-stage oocytes can be manipulated, by microinjection, for example, before maturation begins. Add 250/xM dibutyryl cyclic adenosine monophosphate or 100/~M 3-isobutyl-l-methylxanthine to the maturation medium to maintain meiotic arrest while manipulating the oocytes, then wash the complexes in maturation medium and proceed with oocyte maturation as described above. 8-~° E x p e c t e d Results
The embryonic developmental competence of mouse oocytes matured in oitro will vary according to the age of the donor mice,ll the hormonal
status of the mice donating the GV-stage oocytes, ~2'~3 FSH treatment of the maturing oocytes in vitro, ~1 and the presence or absence of serum in the oocyte maturation medium.7'14 The effect of the genotype of the oocyte donor has not been assessed. Nevertheless, when PMSG-primed (C57BL/ 6J × SJL/J)F 1 mice 24 to 26 days old provide the GV-stage oocytes for maturation in medium supplemented with FBS, the frequency of fertilization and cleavage to the 2-cell stage should be 75 to 95%. Moreover, 65 to 4 M. DeFelici, A. Salustri, and G. Siracusa, Gamete Res. 12, 227 (1985). 5 j. G. Gianfortoni and B. J. Gulyas, Gamete Res. 11, 59 (1985). 6 S. M. Downs, A. C. Schroeder, and J. J. Eppig, Gamete Res. 15, 115 (1986). 7 A. C. Schroeder, R. M. Schultz, G. S. Kopf, F. R. Taylor, R. B. Becker, and J. J. Eppig, Biol. Reprod. 43, 891 (1990). 8 W. K. Cho, S. Stern, and J. D. Biggers, J. Exp. Zool. 187, 383 (1974). 9 C. Magnusson and T. Hillensjo, J. Exp. Zool. 201, 138 (1977). 10 S. M. Downs, A. C. Schroeder, and J. J. Eppig, Gamete Res. 15, 305 (1986). u j. j. Eppig and A. C. Schroeder, Biol. Reprod. 41, 268 (1989). 12 j. j. Eppig, A. C. Schroeder, and M. J. O'Brien, J. Reprod. Fertil. 95, 119 (1992). 13 A. C. Schroeder and J. J. Eppig, Gamete Res. 24, 81 (1989). 14j. j. Eppig, K. Wigglesworth, and M. J. O'Brien, Mol. Reprod. Dev. 32, 33 (1992).
[5]
OOCYTE ISOLATION AND CULTURE
81
85% of the 2-cell stage embryos should develop to the expanded blastocyst stage. Between 30 and 45% of the 2-cell stage embryos transferred to the oviducts of foster mothers should develop to live offspring.
Isolation, Growth, and Maturation of Growing, Meiotically Incompetent Germinal Vesicle-Stage Oocytes
Animals Large numbers of oocyte-granulosa cell complexes can be isolated from the preantral follicles of 12-day-old mice. Use of the immature mice provides ovaries that are readily dissociated with collagenase and also relatively homogeneous populations of preantral follicles.
Isolation and Collection of Oocyte-Granulosa Cell Complexes of Preantral Follicles Ovaries from 5 to 15 mice are immersed in 3.5 ml of culture medium containing 1 to 2 mg/ml crude collagenase (Worthington type CLS-1, Freehold, N J, for example) and 0.02 mg/ml DNase (sterilized by filtration) and then are placed on a slide warming tray maintained at 35° and covered with a plexiglass box that is constantly flushed with 5-5-90 gas. After about 10 min, the ovaries are vigorously drawn in and out of an Eppendorf micropipette set at 1.0 ml and equipped with a sterile blue tip until the ovaries begin to dissociate. The ovaries are returned to the warming tray for 10-min intervals, at which times the dissociating ovaries are repeatedly drawn in and out of a micropipette set at 0.1 ml and equipped with a sterile yellow tip. This process is repeated until the ovaries are completely dissociated into individual oocyte-granulosa cell complexes. This is usually completed within 40 min. The complexes are washed by swirling them together in the center of the petri dish, drawing off the cloud of debris that overhangs the settling complexes, transferring the complexes to another petri dish containing 3.0 ml of medium, and repeating this process until the preparation is free of ovarian debris, usually four or five transfers. This procedure results in a preparation such as that shown in Fig. 2. The complexes each consist of an oocyte, which is in mid-growth phase and incompetent of undergoing GV breakdown without further development, surrounded by one to three layers of granulosa cells. Most of the primitive theca layers and the basal lamina are removed by the collagenase digestion.
82
GERM CELLS AND EMBRYOS
[5]
FI6. 2. Photomicrograph of preantral follicles produced by dissociation of the ovaries of 12-day-old mice using collagenase/DNase. Bar: 100/~m.
Oocyte Growth The oocyte-granulosa cell complexes are distributed to the surface of either Transwell-COL membranes (Costar, Cambridge, MA; 3.0/zm pore size) or a rat tail collagen matrix prepared in the following way. 15'16
Preparation of Rat Tail Collagen 1. Tails obtained from rats can be frozen until needed for preparation of collagen. They are sterilized overnight in 70% (v/v) ethanol immediately before use. 2. Dissect out tendons using two pairs of artery forceps. Starting from the tip of the tail clamp one end with artery forceps and clamp approximately 3 cm away from the other pair. After bending and pulling both pairs of forceps, the tendons will come loose from the skin. Cut the tendons loose and place them in a small beaker with 70% ethanol. 3. Wash tendons in sterile distilled water and blot dry with sterile filter paper. 15 R. I. Ehrmann and G. O. Gey, J. Natl. Cancer Inst. 16, 1375 (1956). 16 M. Chambard, J. Gabrion, and J. Mauchamp, J. Cell Biol. 91, 157 (1981).
[5]
OOCYTE ISOLATION AND CULTURE
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4. Weigh tendons and place them in a large bottle with a sterile magnetic stir bar. 5. Add 100 ml of 1 : 1000 acetic acid-water per gram of tendons. 6. Stir tendons on a magnetic stirring plate in a cold room for 48 hr. 7. Transfer collagen to 50-ml centrifuge tubes and centrifuge at 4000 rpm at 4° for 60 min. 8. Dialyze the collagen solution against a 10-fold volume of 1 : 1000 acetic acid-water 3 times for 24 hr each time. 9. Transfer collagen to new 50-ml centrifuge tubes on ice. 10. Store collagen in a refrigerator or freezer.
Preparation of Collagen Gel Matrix for Culture 1. Keep all components on ice. Mix the following in order: 1.6 ml of rat tail collagen, 0.2 ml Dulbecco's phosphate-buffered saline (PBS), and 0.2 ml of 10 × culture medium. 2. Add 70 to 100/zl of an ice-cold 0.5 M NaOH solution. Each lot of collagen is a little different, so the exact amount should be determined. The mixture should be homogeneous and pink in color. 3. Using a micropipette equipped with a sterile blue tip, add 0.250 ml of the collagen solution to the bottom of a well in a 24-well tissue culture cluster dish and distribute evenly. Keep cold during this process. 4. Set the culture dish on the slide warmer and allow the collagen to gel (>15 min). 5. Overlay the collagen gel matrix with about 1 ml of PBS and change every 5 min for 30 min. Add and remove the solutions very gently. 6. Overlay the collagen gel matrix with culture medium and incubate on the warming tray for 5 to 10 min. 7. Discard the medium and replace with 1 ml of fresh culture medium and distribute the oocyte-granulosa cell complexes. Cultures are incubated at 37° in modular incubation chambers flushed with 5-5-90 gas and fed by exchanging approximately one-half of the medium with fresh medium every second day.
Maturation of in Vitro Grown Oocytes After culturing the oocyte-granulosa cell complexes for 10 to 14 days, remove them from the surface of the Transwell-COL membrane or collagen gel matrix. This is done by sharply snapping the side of the dish with a fingernail to jolt the complexes free of the substratum. The complexes are collected with a drawn glass micropipette and matured as described above. After maturation, wash the complexes in the medium used for fertilization and inseminate immediately.
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GERM CELLS AND EMBRYOS
[6]
Expected Results When oocyte-granulosa cell complexes are isolated from the preantral follicles of 12-day-old (C57BL/6J × SJL/J)F 1 mice and cultured for 10 days in FBS-supplemented medium, it should be anticipated that 60 to 80% of the oocytes will acquire competence to mature. Of the mature eggs, 50 to 60% will cleave to the 2-cell stage after insemination, and 55 to 70% of the 2-cell stage embryos will develop to the expanded blastocyst stage. Between 10 and 15% of the 2-cell stage embryos transferred to the oviducts of foster mothers should develop to live offspring. The oocyte-granulosa cell complexes of preantral follicles can also be grown in serum-free medium supplemented with 1 mg/ml fetuin, 5/zg/ml insulin, 5 /zg/ml transferrin, and 5 ng/ml selenium (ITS; Collaborative Research, Bedford, MA). Under these conditions, fewer oocytes competent of subsequent development to the blastocyst stage are produced.14 These results suggest that serum-born growth factors are important for optimal oocyte development.
[6] Purification, C u l t u r e , a n d F r a c t i o n a t i o n of S p e r m a t o g e n i c Cells
By ANTHONYR. BELLVI~ Introduction The adult mammalian testis contains multiple populations of somatic and germinal cells that differ markedly in their stages of growth and differentiation. Germ cells, the major cell type of the testis, undergo a complex and fascinating process of differentiation. 1The process, spermatogenesis, starts with the renewal of the rare spermatogonial stem cells (A0/As), followed by the proliferative sequence of types Apr, Aal, AI_ 4 , intermediate, and type B spermatogonia (Fig. 1). The latter form primary spermatocytes, which enter a prolonged meiotic prophase to effect pairing of homologous chromosomes, genetic recombination, and, with the two reduction divisions, random segregation of chromosomes to yield haploid spermatids. Then, during spermiogenesis, the spermatids undergo a remarkable sequence of differentiation that includes condensing the chromatin, reorganizing and shaping the nucleus, forming an acrosome replete with enzymes to aid fertilization, and assembling a tail with its microtubule I A. R. Bellv6,
Oxford Reo. Reprod. Biol. 1, 159 (1979).
METHODS IN ENZYMOLOGY, VOL. 225
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