MOLECULAR REPRODUCTION AND DEVELOPMENT 27:235-243 (1990)
Protein Synthesis Inhibitors Prevent Both Spontaneous and Hormone-Dependent Maturation of Isolated Mouse Oocytes STEPHEN M. DOWNS Biology Department and Biological and Biomedical Research Institute, Marquette University, Milwaukee, Wisconsin The present study was carried ABSTRACT out to examine the role of protein synthesis in mouse oocyte maturation in vitro. In the first part of this study, the effects of cycloheximide (CX)were tested on spontaneous meiotic maturation when oocytes were cultured in inhibitor-free medium. CX reversibly suppressed maturation of oocytes as long as maturation was either initially prevented by the phosphodiesterase inhibitor, 3-isobutyl-1 -methylxanthine (IBMX), or delayed by follicle-stimulating hormone (FSH).In the second part of this study, the actions of protein synthesis inhibitors were tested on hormone-induced maturation. CEO were maintained in meiotic arrest for 21-22 h with hypoxanthine, and germinal vesicle breakdown (GVB) was induced with follicle-stimulating hormone (FSH). Three different protein synthesis inhibitors [CX, emetine (EM), and puromycin (PUR)] each prevented the stimulatory action of FSH on GVB in a dose-dependent fashion. This was accompanied by a dose-dependent suppression of 3H-leucine incorporation by oocyte-cumulus cell complexes. The action of these inhibitors on FSH- and epidermal growth factor (EGF)-induced GVB was next compared. All three drugs lowered the frequency of GVB in the FSH-treated groups, below even that of hypoxanthine); the drugs the controls (drug maintained meiotic arrest at the control frequencies in the EGF-treated groups. Puromycin aminonucleoside, an analog of PUR with no inhibitory action on protein synthesis, had no effect. The three inhibitors also suppressed the stimulatory action of FSH on oocyte maturation when meiotic arrest was maintained with the CAMP analog, dbcAMP. In a time course experiment, it was determined that CX could prevent FSH-induced maturation if added as late as 3 h after the onset of culture, but the drug was less effective in EGF-treated cultures. It is concluded that a protein with a rapid turnover rate is involved in the spontaneous maturation of mouse oocytes and that de novo protein synthesis is a requirement for hormone induction of GVB in vitro.
INTRODUCTION Knowledge concerning the metabolic requirements for the resumption of meiotic maturation in mammalian oocytes is limited. Poorly understood is the role protein synthesis plays in this physiological process. A number of laboratories have demonstrated pronounced changes in the protein synthetic profiles of mammalian oocytes during meiotic maturation (Golbus and Stein, 1976; Schultz and Wassarman, 1977a,b; McGaughey and Van Blerkom, 1977; Mangia and Canipari, 1977; Warnes et al., 1977; Van Blerkom and McGaughey, 1978). In addition, protein synthesis has been shown to be a requirement for the spontaneous maturation of cow (Hunter and Moor, 1987), pig (Fulka et al., 19861, and sheep (Moor and Crosby, 1986) oocytes in vitro, and treatment of sheep oocytes with cycloheximide (CX) reversibly suppressed the appearance of a protein correlated with GVB (Moor and Crosby, 1986). However, translation inhibitors have no effect on spontaneous GVB in cultured mouse oocytes, even though nuclear maturation to metaphase I1 is suppressed (Stern et al., 1972; Golbus and Stein, 1976; Wassarman et al., 1976; Schultz and Wassarman, 1977a; Fulka et al., 1986). It is possible that the time course of GVB in the various species dictates the sensitivity of the oocyte to protein synthesis inhibitors. Accordingly, in rodent species the oocyte would not be sensitive to protein synthesis inhibitors because of the rapid maturation kinetics; i.e., the oocytes become committed to undergo germinal vesicle breakdown (GVB) before the proteins involved are turned over. Ekholm and Magnusson (1979) have presented evidence in the rat to support this idea. When rat oocytes were cultured in medium supplemented with puromycin (PUR), the drug had no effect on GVB. However, if the oocytes were initially incubated in medium containing dbcAMP (which maintained meiotic arrest) plus PUR and then transferred to medium containing
Key Words: Cycloheximide, Meiotic maturation,
Received October 24, 1989; accepted May 22, 1990. Address reprint requests to Stephen M. Downs, Biology Department, 530 N 15th Street, Marquette University, Milwaukee, WI 53233.
+
Emetine, Puromycin
O 1990 WILEY-LISS, INC.
236
S.M. DOWNS
only PUR, the drug prevented maturation, its inhibitory activity directly related to the length of the initial incubation period. In addition, PUR was inhibitory when oocytes were first incubated within their follicles in PUR-containing medium and then isolated from the follicle and cultured in PUR-containing medium. These results indicated that if oocytes were maintained in meiotic arrest in the presence of protein synthesis inhibitors, short-lived proteins involved in maturation would eventually be degraded and, upon return to a permissive medium, the oocytes would not undergo GVB in the absence of new protein synthesis (Ekholm and Magnusson, 1979). The present studies were undertaken to evaluate the role of protein synthesis in mouse oocyte maturation in vitro. To this end, two types of experiments were carried out. In the first series of experiments, a n inhibitory action of CX on spontaneous oocyte maturation was demonstrated when meiosis was initially prevented by IBMX or delayed with FSH. The results of these experiments suggest, in agreement with Ekholm and Magnusson (19791, the participation of short-lived proteins in spontaneous meiotic maturation. The second series of experiments took advantage of the fact that isolated cumulus cell-enclosed mouse oocytes, maintained in meiotic arrest in vitro with HX or dbCAMP,can be stimulated to undergo GVB with FSH or epidermal growth factor (EGF) (Downs et al., 1988). Utilizing this model system, three different protein synthesis inhibitors have been shown to suppress hormone-induced oocyte maturation. The results of these experiments support an essential role for protein synthesis in the meiosis-inducing action of FSH and EGF.
MATERIALS AND METHODS The mice used in this study were (C57BW6J x SJL/ J) F, females, 19-21 days of age. Mice received 5 IU pregnant mare serum gonadotropin (Diosynth, Inc., Chicago, IL) 48 h prior to use. Large antral follicles were punctured with sterile needles and cumulus cellenclosed oocytes (CEO) were collected. Following isolation, CEO were washed through four changes of medium and transferred to the appropriate test medium. Specific collection conditions for each experiment are detailed in the table and figure legends. For experiments involving culture times of 12 h or less, oocytes were cultured in 1.0 or 1.5 ml medium in borosilicate culture tubes with a rubber stopper a t 37°C. For 2122 h cultures, oocytes were incubated in 35 mm Falcon or Corning Petri dishes in 3.0 ml medium. All media were gassed with a humidified mixture of 5% 02,5% COP, and 90% NP. The culture medium used for all experiments was Eagle’s minimum essential medium (MEM) with Earle’s salts and 0.23 mM pyruvate. All media were supplemented with 3 mg/ml lyophilized crystallized bovine serum albumin (ICN ImmunoBiologicals, Lisle, IL).
Each experiment was performed three or more times with at least 50 oocytes cultured per group per experiment. At the end of the culture period, cumulus cells were removed and oocytes were scored for GVB. For statistical analysis of a given experiment, maturation frequencies were subjected to arcsin transformation followed by Student’s t test or ANOVA; when significant differences were indicated by ANOVA, data were further analyzed by Duncan’s multiple range test. Significance was inferred at P
Protein Synthesis Inhibitors Prevent Both Spontaneous and Hormone-Dependent Maturation of Isolated Mouse Oocytes STEPHEN M. DOWNS Biology Department and Biological and Biomedical Research Institute, Marquette University, Milwaukee, Wisconsin The present study was carried ABSTRACT out to examine the role of protein synthesis in mouse oocyte maturation in vitro. In the first part of this study, the effects of cycloheximide (CX)were tested on spontaneous meiotic maturation when oocytes were cultured in inhibitor-free medium. CX reversibly suppressed maturation of oocytes as long as maturation was either initially prevented by the phosphodiesterase inhibitor, 3-isobutyl-1 -methylxanthine (IBMX), or delayed by follicle-stimulating hormone (FSH).In the second part of this study, the actions of protein synthesis inhibitors were tested on hormone-induced maturation. CEO were maintained in meiotic arrest for 21-22 h with hypoxanthine, and germinal vesicle breakdown (GVB) was induced with follicle-stimulating hormone (FSH). Three different protein synthesis inhibitors [CX, emetine (EM), and puromycin (PUR)] each prevented the stimulatory action of FSH on GVB in a dose-dependent fashion. This was accompanied by a dose-dependent suppression of 3H-leucine incorporation by oocyte-cumulus cell complexes. The action of these inhibitors on FSH- and epidermal growth factor (EGF)-induced GVB was next compared. All three drugs lowered the frequency of GVB in the FSH-treated groups, below even that of hypoxanthine); the drugs the controls (drug maintained meiotic arrest at the control frequencies in the EGF-treated groups. Puromycin aminonucleoside, an analog of PUR with no inhibitory action on protein synthesis, had no effect. The three inhibitors also suppressed the stimulatory action of FSH on oocyte maturation when meiotic arrest was maintained with the CAMP analog, dbcAMP. In a time course experiment, it was determined that CX could prevent FSH-induced maturation if added as late as 3 h after the onset of culture, but the drug was less effective in EGF-treated cultures. It is concluded that a protein with a rapid turnover rate is involved in the spontaneous maturation of mouse oocytes and that de novo protein synthesis is a requirement for hormone induction of GVB in vitro.
INTRODUCTION Knowledge concerning the metabolic requirements for the resumption of meiotic maturation in mammalian oocytes is limited. Poorly understood is the role protein synthesis plays in this physiological process. A number of laboratories have demonstrated pronounced changes in the protein synthetic profiles of mammalian oocytes during meiotic maturation (Golbus and Stein, 1976; Schultz and Wassarman, 1977a,b; McGaughey and Van Blerkom, 1977; Mangia and Canipari, 1977; Warnes et al., 1977; Van Blerkom and McGaughey, 1978). In addition, protein synthesis has been shown to be a requirement for the spontaneous maturation of cow (Hunter and Moor, 1987), pig (Fulka et al., 19861, and sheep (Moor and Crosby, 1986) oocytes in vitro, and treatment of sheep oocytes with cycloheximide (CX) reversibly suppressed the appearance of a protein correlated with GVB (Moor and Crosby, 1986). However, translation inhibitors have no effect on spontaneous GVB in cultured mouse oocytes, even though nuclear maturation to metaphase I1 is suppressed (Stern et al., 1972; Golbus and Stein, 1976; Wassarman et al., 1976; Schultz and Wassarman, 1977a; Fulka et al., 1986). It is possible that the time course of GVB in the various species dictates the sensitivity of the oocyte to protein synthesis inhibitors. Accordingly, in rodent species the oocyte would not be sensitive to protein synthesis inhibitors because of the rapid maturation kinetics; i.e., the oocytes become committed to undergo germinal vesicle breakdown (GVB) before the proteins involved are turned over. Ekholm and Magnusson (1979) have presented evidence in the rat to support this idea. When rat oocytes were cultured in medium supplemented with puromycin (PUR), the drug had no effect on GVB. However, if the oocytes were initially incubated in medium containing dbcAMP (which maintained meiotic arrest) plus PUR and then transferred to medium containing
Key Words: Cycloheximide, Meiotic maturation,
Received October 24, 1989; accepted May 22, 1990. Address reprint requests to Stephen M. Downs, Biology Department, 530 N 15th Street, Marquette University, Milwaukee, WI 53233.
+
Emetine, Puromycin
O 1990 WILEY-LISS, INC.
236
S.M. DOWNS
only PUR, the drug prevented maturation, its inhibitory activity directly related to the length of the initial incubation period. In addition, PUR was inhibitory when oocytes were first incubated within their follicles in PUR-containing medium and then isolated from the follicle and cultured in PUR-containing medium. These results indicated that if oocytes were maintained in meiotic arrest in the presence of protein synthesis inhibitors, short-lived proteins involved in maturation would eventually be degraded and, upon return to a permissive medium, the oocytes would not undergo GVB in the absence of new protein synthesis (Ekholm and Magnusson, 1979). The present studies were undertaken to evaluate the role of protein synthesis in mouse oocyte maturation in vitro. To this end, two types of experiments were carried out. In the first series of experiments, a n inhibitory action of CX on spontaneous oocyte maturation was demonstrated when meiosis was initially prevented by IBMX or delayed with FSH. The results of these experiments suggest, in agreement with Ekholm and Magnusson (19791, the participation of short-lived proteins in spontaneous meiotic maturation. The second series of experiments took advantage of the fact that isolated cumulus cell-enclosed mouse oocytes, maintained in meiotic arrest in vitro with HX or dbCAMP,can be stimulated to undergo GVB with FSH or epidermal growth factor (EGF) (Downs et al., 1988). Utilizing this model system, three different protein synthesis inhibitors have been shown to suppress hormone-induced oocyte maturation. The results of these experiments support an essential role for protein synthesis in the meiosis-inducing action of FSH and EGF.
MATERIALS AND METHODS The mice used in this study were (C57BW6J x SJL/ J) F, females, 19-21 days of age. Mice received 5 IU pregnant mare serum gonadotropin (Diosynth, Inc., Chicago, IL) 48 h prior to use. Large antral follicles were punctured with sterile needles and cumulus cellenclosed oocytes (CEO) were collected. Following isolation, CEO were washed through four changes of medium and transferred to the appropriate test medium. Specific collection conditions for each experiment are detailed in the table and figure legends. For experiments involving culture times of 12 h or less, oocytes were cultured in 1.0 or 1.5 ml medium in borosilicate culture tubes with a rubber stopper a t 37°C. For 2122 h cultures, oocytes were incubated in 35 mm Falcon or Corning Petri dishes in 3.0 ml medium. All media were gassed with a humidified mixture of 5% 02,5% COP, and 90% NP. The culture medium used for all experiments was Eagle’s minimum essential medium (MEM) with Earle’s salts and 0.23 mM pyruvate. All media were supplemented with 3 mg/ml lyophilized crystallized bovine serum albumin (ICN ImmunoBiologicals, Lisle, IL).
Each experiment was performed three or more times with at least 50 oocytes cultured per group per experiment. At the end of the culture period, cumulus cells were removed and oocytes were scored for GVB. For statistical analysis of a given experiment, maturation frequencies were subjected to arcsin transformation followed by Student’s t test or ANOVA; when significant differences were indicated by ANOVA, data were further analyzed by Duncan’s multiple range test. Significance was inferred at P
