MOLECULAR REPRODUCTION AND DEVELOPMENT 3359-66 (1992)
Effects of Protein Synthesis on Maturation, Sperm Penetration, and Pronuclear Development in Porcine Oocytes JIANCHI DING,' ROBERT M. MOOR:
AND
GEORGE R. FOXCROFT'
'Department of Animal Science, Faculty of Agriculture and Forestry, University of Alberta, Edmonton, Alberta, Canada; 'Department of Molecular Embryology, AFRC Institute of Animal Physiology and Genetic Research, Babraham, Cambridge, United Kingdom
ABSTRACT In vitro matured porcine oocytes were used to test the importance of protein synthesis for sperm penetration, the second meiotic division, and pronuclear development. Experiments were carried out to measure rates of protein synthesis in the presence of protein synthesis inhibitors (35 p M or 350 pM cycloheximide or a combination of inhibitors) (study 1);to test for sperm penetration and pronuclear development when protein synthesis was inhibited during fertilization (study 2); to test for oocyte meiosis, sperm penetration, and female and male pronuclear development when protein synthesis was inhibited during maturation (oocyte maturation in vitro with addition of inhibitor at 0, 24, or 36 hr of culture) (study 3); and to analyze the changes in the pattern of protein synthesis during these phases. Sperm penetration, oocyte meiosis, and female pronuclear development were not affected by the total inhibition of protein synthesis during fertilization. By contrast, inhibiting protein synthesis during maturation severely impaired the completion of meiosis and pronuclear development. Although inhibition of protein synthesis after 36 hr of maturation culture did not totally block male pronuclear development (MPN), the rate of MPN formation was lower than for controls (52%vs. 72%, P < 0.05). However, protein synthesis was absolutely essential between 24 and 36 hr for the formation of MPN after decondensation. This period of maturation coincided with the dominant phase of protein reprogramming in the Oocyte. Q 1992 Wiley-Liss, Inc.
pothesize t h a t de novo synthesis of proteins during fertilization may not be obligatory for sperm penetration, the second meiotic division, or pronuclear development. The present study was designed first to test this hypothesis by examining whether sperm penetration and subsequent pronuclear development occur in oocytes whose protein synthetic capacity is inhibited during fertilization. Establishing that de novo synthesis of proteins during fertilization is not required led to a second series of experiments to identify the stage during oocyte maturation at which proteins required for activation, sperm decondensation, and pronuclear formation are synthesized.
MATERIALS AND METHODS Preparation of Oocytes Ovaries were obtained from slaughtered gilts a t a local abattoir and were transported to the laboratory within 40 min in a polystyrene box to prevent major changes in temperature. Follicles were dissected from the ovary, and oocyte-cumulus-granulosa cell complexes (oocyte complexes) were harvested from healthy follicles with a diameter of 3-6 mm. Dissection was carried out in medium 199 with 25 mM Hepes (Earle's salts; Gibco, Grand Island, NY) containing 10% heattreated newborn calf serum (Gibco) at a temperature of 24-26°C. The dissecting procedure was completed within 2.5 hr. Groups of 10-15 randomly selected oocyte complexes were incubated in-35 mm plastic petri dishes containing 2 ml medium 199 supplemented with Key Words: Fertilization, Pig Oocyte, Meiosis 10%fetal calf serum (FCS; Gibco), 100 pg/ml glutamine (BDH; Sigma, St. Louis, MO), 70 pg/ml L-ascorbic acid INTRODUCTION (BDH; Sigma), 35 pg/ml insulin (BDH; Sigma), and De novo protein synthesis is essential for resumption gonadotrophins' (2.5 pg/ml NTADDK-oLH-26, AFPof meiosis of ungulate oocytes (Hunter and Moor, 1987; 5551b; 2.5 pg/ml USDA-pFSH-B-1, AFP-5600) and Moor and Crosby, 1986; Fulka et al., 1986). Whether i t prolactin (20 ng/ml USDA-pprl-B-1, AFP-5000). TWO is also essential for fertilization and pronuclear devel- everted follicle shells (4-6 mm diameter) were added to opment has not been ascertained in domestic species. Biochemical results showed that proteins synthesized before fertilization undergo changes similar to those of proteins synthesized during fertilization (Ding et al., Received January 30, 1992; accepted March 2,1992. reprint requests to Jianchi Ding, Department of Animal Sci1992). These findings suggest, therefore, that protein Address ence, University of Alberta, 310 Agriculture-Forestry Building, Edchanges associated with fertilization occur primarily a t monton, Alberta, Canada T6G 2P5. the posttranslational modification level using precur- 'Gonadotropins and prolactin were gifts from the National Institute of sors accumulated during maturation. From this we hy- Diabetes and Digestive and Kidney Diseases (NIDDK).
0 1992 WILEY-LISS, INC.
60
J. DING ET AL.
each dish and cocultured with the oocytes. Culture was carried out with gentle agitation under a n atmosphere of 5% C 0 2 in air a t 39°C for 47 1hr.
*
Sperm Preparation and In Vitro Fertilization (IVF) and Nuclear Examination Preparation of spermatozoa. Four mature Landrace boars were used €or supplying semen. For each IVF study, sperm-rich fractions were obtained from two boars by a gloved-hand method, After removing the gel fraction, the two semen samples were mixed (1:l)and held a t 20°C for 16 h r (Cheng et al., 1986). Spermatozoa were washed and preincubated for 90 min before being cocultured with oocytes as described by Ding et al. (1992). In vitro fertilization. Cumulus-enclosed oocytes matured for 47 _C 1 h r in vitro were incubated with preincubated boar spermatozoa at a concentration of 5 x lo5 cellsiml in B.O. medium (Brackett and Oliphant, 1975; all the chemicals were from Sigma BDH; BSA was fraction V.) for 10 hr. Examination of nuclear status. After 47 I1 h r maturation culture or 10 h r after insemination, oocytes were denuded of cumulus cells, mounted on a slide using a whole mount technique, and fixed for 48 h r in ethano1:acetic acid (3:l). The nuclear status of oocytes was examined under a phase-contrast microscope after staining with 1% lacmoid in 45% acetic acid solution. The stage of nuclear maturation was identified basically according to Hunter and Polge (19661, and the nuclear status of oocytes after fertilization was identified a s described previously (Ding e t al., 1992). Some unusual nuclear configurations are shown in Figure 1. Those penetrated oocytes with two or more polar bodies (Pb) and one full-sized female pronucleus were considered as having completed the second meiosis and normal female pronuclear development. Finally, those penetrated oocytes with full-sized male pronucleusi pronuclei were considered to have undergone normal male pronuclear development.
cover slip was carefully removed and oocytes were individually recovered from the slide and placed in groups according to their nuclear configuration. A group of one to three radiolabelled oocytes with the same nuclear configuration was washed briefly in 10 mM Tris-0.1 mM EDTA solution, transferred in a small volume (1,000 Ci/mM; Amersham) a t 39°C for 3 hr, in the presence or absence (control) of a chain-elongation inhibitor (cycloheximide a t 35 or 350 pM) or a combination of inhibitors of chain elongation (35 pM cycloheximide), ribosomal subunit function (0.1 pM emetine), and a n inducer of premature peptide chain release (20 pM puromycin). Following labelling, the oocytes were denuded of cumulus cells and washed four times before transfer of single oocytes in 2 pl of 10 mM Tris-0.1 mM EDTA to 0.5 ml microtubes for lyophilization and subsequent storage a t -70°C. Assays for nonspecific binding were carried out by taking, before labelling of oocytes, 2 pl of [35S]methionine-containing medium and thereafter treating these blank samples in the same manner as those containing eggs. Lyophilized samples were prepared for further analysis by adding 20 pl of gel SDS buffer (O’Farrell, 19751, followed by three cycles of freezing and heating. A 5 pl aliquot of each sample was used to measure the total radioactivity as a parameter Measurement of Protein Synthesis of total uptake of [35Slmethionine by the oocyte, and Freshly prepared oocyte complexes (see above) were another 5 ~1 aliquot was used to determine incorporalabelled with [35S]methionine at a final concentration tion of [35S]methionine into trichloroacetic acid (TCA)of 500 p.Ci/ml (specific activity >1,000 CiimM; Amer- precipitable material as described by Van Blerkom sham, Amersham, England) for 3 h r a t 39°C (see Moor (1978). In addition to the blank samples (radiolabelled et al., 1981) a t various times during in vitro maturation medium), nonspecific binding was further analyzed by (for details, see legend to Fig. 3). After incubation, loading SDS gels with the same number of TCA-precipoocytes were washed with [35S]methionine-free label- itable counts from untreated controls and from inhibiling medium and transiently cultured (15-20 min) in tor-treated groups. Pooling of a large number of sammedium containing 10 pgiml DAPI (4,6-diamidino-2- ples of eggs subjected to the same inhibitor regimen phenylindole; Sigma), a DNA fluorescent stain mate- was necessary to obtain adequate counts for these exrial. Oocytes thereafter were denuded of cumulus cells periments. and loaded, in a small amount of the same medium (2-3 pl), into small individual compartments on a slide and Statistical Analysis covered gently with a coverslip to prevent quick evaporation of the medium. Both support of the coverslip and Data in study 1were subjected to a one-way analysis separation of individual oocytes were achieved using of variance, and percentage data from studies 2 and 3 thin lines of Vaseline. Nuclear status was determined were subjected to a two-way variance analysis of a ranunder a fluorescent microscope, following which the dom block design after arcsine transformation. Multi-
MATURATION OF PIG OOCYTES
61
TABLE 1. Effect of Inhibiting Protein Synthesis After In Vitro Maturation on Subsequent Sperm Penetration and on Male (MPN)and Female (FPN) Pronuclear Formation* Treatments 35 pM cyclo. 350 pM cyclo. Inhib. mix. Control No. replicates 5 5 5 5 No. MI1 oocytes 62 69 77 58 Penetrated MI1 oocytes No. 57 50 69 53 % (mean k s.e.) 92.9 f 7.2a 74.3 f 14.7a 87.9 k 8.Eia 92.5 k 4.8" Penetrated oocytes with 2 Pb 1 FPN No. 47 41 56 40 % (mean f s.e.) 81.8 t 5.ga 83.8 f 7.5a 75.0 k 6.3a 83.4 t 5.9" Penetrated oocytes with MPN 42 37 45 34 No. % (mean f s.e.) 73.1 k 2.8" 72.5 3.4a 68.5 k 7.0" 65.3 k 3.0a *In vitro-matured oocytes were cocultured with in vitro-capacitated boar spermatozoa for 10 hr in B.O. medium containing 1)35 pM cycloheximide (cyclo.),2) 350 pM cyclo., and 3) combined inhibitors (inhib. mix.), and 4) a control receiving no protein synthesis inhibitors. Fixed oocytes were stained with 1% lacmoid and examined under a phase-contrast microscope. "With different subscripts within a row: P < 0.05.
+
*
ple comparisons of means were made by Student-Newman-Keul's tests.
than that suggested by the corrected TCA results. The apparent cycloheximide-resistant protein ran as a faint smear on the gel, represented by densitometry readings RESULTS of
Effects of Protein Synthesis on Maturation, Sperm Penetration, and Pronuclear Development in Porcine Oocytes JIANCHI DING,' ROBERT M. MOOR:
AND
GEORGE R. FOXCROFT'
'Department of Animal Science, Faculty of Agriculture and Forestry, University of Alberta, Edmonton, Alberta, Canada; 'Department of Molecular Embryology, AFRC Institute of Animal Physiology and Genetic Research, Babraham, Cambridge, United Kingdom
ABSTRACT In vitro matured porcine oocytes were used to test the importance of protein synthesis for sperm penetration, the second meiotic division, and pronuclear development. Experiments were carried out to measure rates of protein synthesis in the presence of protein synthesis inhibitors (35 p M or 350 pM cycloheximide or a combination of inhibitors) (study 1);to test for sperm penetration and pronuclear development when protein synthesis was inhibited during fertilization (study 2); to test for oocyte meiosis, sperm penetration, and female and male pronuclear development when protein synthesis was inhibited during maturation (oocyte maturation in vitro with addition of inhibitor at 0, 24, or 36 hr of culture) (study 3); and to analyze the changes in the pattern of protein synthesis during these phases. Sperm penetration, oocyte meiosis, and female pronuclear development were not affected by the total inhibition of protein synthesis during fertilization. By contrast, inhibiting protein synthesis during maturation severely impaired the completion of meiosis and pronuclear development. Although inhibition of protein synthesis after 36 hr of maturation culture did not totally block male pronuclear development (MPN), the rate of MPN formation was lower than for controls (52%vs. 72%, P < 0.05). However, protein synthesis was absolutely essential between 24 and 36 hr for the formation of MPN after decondensation. This period of maturation coincided with the dominant phase of protein reprogramming in the Oocyte. Q 1992 Wiley-Liss, Inc.
pothesize t h a t de novo synthesis of proteins during fertilization may not be obligatory for sperm penetration, the second meiotic division, or pronuclear development. The present study was designed first to test this hypothesis by examining whether sperm penetration and subsequent pronuclear development occur in oocytes whose protein synthetic capacity is inhibited during fertilization. Establishing that de novo synthesis of proteins during fertilization is not required led to a second series of experiments to identify the stage during oocyte maturation at which proteins required for activation, sperm decondensation, and pronuclear formation are synthesized.
MATERIALS AND METHODS Preparation of Oocytes Ovaries were obtained from slaughtered gilts a t a local abattoir and were transported to the laboratory within 40 min in a polystyrene box to prevent major changes in temperature. Follicles were dissected from the ovary, and oocyte-cumulus-granulosa cell complexes (oocyte complexes) were harvested from healthy follicles with a diameter of 3-6 mm. Dissection was carried out in medium 199 with 25 mM Hepes (Earle's salts; Gibco, Grand Island, NY) containing 10% heattreated newborn calf serum (Gibco) at a temperature of 24-26°C. The dissecting procedure was completed within 2.5 hr. Groups of 10-15 randomly selected oocyte complexes were incubated in-35 mm plastic petri dishes containing 2 ml medium 199 supplemented with Key Words: Fertilization, Pig Oocyte, Meiosis 10%fetal calf serum (FCS; Gibco), 100 pg/ml glutamine (BDH; Sigma, St. Louis, MO), 70 pg/ml L-ascorbic acid INTRODUCTION (BDH; Sigma), 35 pg/ml insulin (BDH; Sigma), and De novo protein synthesis is essential for resumption gonadotrophins' (2.5 pg/ml NTADDK-oLH-26, AFPof meiosis of ungulate oocytes (Hunter and Moor, 1987; 5551b; 2.5 pg/ml USDA-pFSH-B-1, AFP-5600) and Moor and Crosby, 1986; Fulka et al., 1986). Whether i t prolactin (20 ng/ml USDA-pprl-B-1, AFP-5000). TWO is also essential for fertilization and pronuclear devel- everted follicle shells (4-6 mm diameter) were added to opment has not been ascertained in domestic species. Biochemical results showed that proteins synthesized before fertilization undergo changes similar to those of proteins synthesized during fertilization (Ding et al., Received January 30, 1992; accepted March 2,1992. reprint requests to Jianchi Ding, Department of Animal Sci1992). These findings suggest, therefore, that protein Address ence, University of Alberta, 310 Agriculture-Forestry Building, Edchanges associated with fertilization occur primarily a t monton, Alberta, Canada T6G 2P5. the posttranslational modification level using precur- 'Gonadotropins and prolactin were gifts from the National Institute of sors accumulated during maturation. From this we hy- Diabetes and Digestive and Kidney Diseases (NIDDK).
0 1992 WILEY-LISS, INC.
60
J. DING ET AL.
each dish and cocultured with the oocytes. Culture was carried out with gentle agitation under a n atmosphere of 5% C 0 2 in air a t 39°C for 47 1hr.
*
Sperm Preparation and In Vitro Fertilization (IVF) and Nuclear Examination Preparation of spermatozoa. Four mature Landrace boars were used €or supplying semen. For each IVF study, sperm-rich fractions were obtained from two boars by a gloved-hand method, After removing the gel fraction, the two semen samples were mixed (1:l)and held a t 20°C for 16 h r (Cheng et al., 1986). Spermatozoa were washed and preincubated for 90 min before being cocultured with oocytes as described by Ding et al. (1992). In vitro fertilization. Cumulus-enclosed oocytes matured for 47 _C 1 h r in vitro were incubated with preincubated boar spermatozoa at a concentration of 5 x lo5 cellsiml in B.O. medium (Brackett and Oliphant, 1975; all the chemicals were from Sigma BDH; BSA was fraction V.) for 10 hr. Examination of nuclear status. After 47 I1 h r maturation culture or 10 h r after insemination, oocytes were denuded of cumulus cells, mounted on a slide using a whole mount technique, and fixed for 48 h r in ethano1:acetic acid (3:l). The nuclear status of oocytes was examined under a phase-contrast microscope after staining with 1% lacmoid in 45% acetic acid solution. The stage of nuclear maturation was identified basically according to Hunter and Polge (19661, and the nuclear status of oocytes after fertilization was identified a s described previously (Ding e t al., 1992). Some unusual nuclear configurations are shown in Figure 1. Those penetrated oocytes with two or more polar bodies (Pb) and one full-sized female pronucleus were considered as having completed the second meiosis and normal female pronuclear development. Finally, those penetrated oocytes with full-sized male pronucleusi pronuclei were considered to have undergone normal male pronuclear development.
cover slip was carefully removed and oocytes were individually recovered from the slide and placed in groups according to their nuclear configuration. A group of one to three radiolabelled oocytes with the same nuclear configuration was washed briefly in 10 mM Tris-0.1 mM EDTA solution, transferred in a small volume (1,000 Ci/mM; Amersham) a t 39°C for 3 hr, in the presence or absence (control) of a chain-elongation inhibitor (cycloheximide a t 35 or 350 pM) or a combination of inhibitors of chain elongation (35 pM cycloheximide), ribosomal subunit function (0.1 pM emetine), and a n inducer of premature peptide chain release (20 pM puromycin). Following labelling, the oocytes were denuded of cumulus cells and washed four times before transfer of single oocytes in 2 pl of 10 mM Tris-0.1 mM EDTA to 0.5 ml microtubes for lyophilization and subsequent storage a t -70°C. Assays for nonspecific binding were carried out by taking, before labelling of oocytes, 2 pl of [35S]methionine-containing medium and thereafter treating these blank samples in the same manner as those containing eggs. Lyophilized samples were prepared for further analysis by adding 20 pl of gel SDS buffer (O’Farrell, 19751, followed by three cycles of freezing and heating. A 5 pl aliquot of each sample was used to measure the total radioactivity as a parameter Measurement of Protein Synthesis of total uptake of [35Slmethionine by the oocyte, and Freshly prepared oocyte complexes (see above) were another 5 ~1 aliquot was used to determine incorporalabelled with [35S]methionine at a final concentration tion of [35S]methionine into trichloroacetic acid (TCA)of 500 p.Ci/ml (specific activity >1,000 CiimM; Amer- precipitable material as described by Van Blerkom sham, Amersham, England) for 3 h r a t 39°C (see Moor (1978). In addition to the blank samples (radiolabelled et al., 1981) a t various times during in vitro maturation medium), nonspecific binding was further analyzed by (for details, see legend to Fig. 3). After incubation, loading SDS gels with the same number of TCA-precipoocytes were washed with [35S]methionine-free label- itable counts from untreated controls and from inhibiling medium and transiently cultured (15-20 min) in tor-treated groups. Pooling of a large number of sammedium containing 10 pgiml DAPI (4,6-diamidino-2- ples of eggs subjected to the same inhibitor regimen phenylindole; Sigma), a DNA fluorescent stain mate- was necessary to obtain adequate counts for these exrial. Oocytes thereafter were denuded of cumulus cells periments. and loaded, in a small amount of the same medium (2-3 pl), into small individual compartments on a slide and Statistical Analysis covered gently with a coverslip to prevent quick evaporation of the medium. Both support of the coverslip and Data in study 1were subjected to a one-way analysis separation of individual oocytes were achieved using of variance, and percentage data from studies 2 and 3 thin lines of Vaseline. Nuclear status was determined were subjected to a two-way variance analysis of a ranunder a fluorescent microscope, following which the dom block design after arcsine transformation. Multi-
MATURATION OF PIG OOCYTES
61
TABLE 1. Effect of Inhibiting Protein Synthesis After In Vitro Maturation on Subsequent Sperm Penetration and on Male (MPN)and Female (FPN) Pronuclear Formation* Treatments 35 pM cyclo. 350 pM cyclo. Inhib. mix. Control No. replicates 5 5 5 5 No. MI1 oocytes 62 69 77 58 Penetrated MI1 oocytes No. 57 50 69 53 % (mean k s.e.) 92.9 f 7.2a 74.3 f 14.7a 87.9 k 8.Eia 92.5 k 4.8" Penetrated oocytes with 2 Pb 1 FPN No. 47 41 56 40 % (mean f s.e.) 81.8 t 5.ga 83.8 f 7.5a 75.0 k 6.3a 83.4 t 5.9" Penetrated oocytes with MPN 42 37 45 34 No. % (mean f s.e.) 73.1 k 2.8" 72.5 3.4a 68.5 k 7.0" 65.3 k 3.0a *In vitro-matured oocytes were cocultured with in vitro-capacitated boar spermatozoa for 10 hr in B.O. medium containing 1)35 pM cycloheximide (cyclo.),2) 350 pM cyclo., and 3) combined inhibitors (inhib. mix.), and 4) a control receiving no protein synthesis inhibitors. Fixed oocytes were stained with 1% lacmoid and examined under a phase-contrast microscope. "With different subscripts within a row: P < 0.05.
+
*
ple comparisons of means were made by Student-Newman-Keul's tests.
than that suggested by the corrected TCA results. The apparent cycloheximide-resistant protein ran as a faint smear on the gel, represented by densitometry readings RESULTS of
