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Quantitative cytochemical study of some enzymatic activities in preovulatory bovine oocytes after in vitro maturation
Acta histochem. (Jena) 95, 89-96 (1993) Gustav Fischer Verlag lena· Stuttgart· New York
Ada
his'o~helRiea
Quantitative cytochemical study of some enzymatic activities in preovulatory bovine oocytes after in vitro maturation Bruno FerrandP), Fausto Cremonesti2), Renato Geiger2), Anna Lange Consiglio l ), Antino CarnevalP), and Franca PorcellP) I)Institute of Anatomy of Domestic Animals and 2)Institute of Veterinary Gynecology and Clinical Obstetrics, University of Milano, Via Celoria 10, 20133 Milano, Italy Accepted 1 June 1993
Summary In the present study quantitative cytochemical assays were used to measure some enzymatic activities in situ in bovine meiotically immature oocytes and oocytes matured in vitro, since the special metabolic activity of the growing oocytes may be a pivotal factor in stabilizing the meiotically arrested oocytes. Modifications of this particular metabolism might destabilize the arrested meiosis. Preovulatory oocytes, mostly at the germinal vesicle stage, were obtained by puncturing follicles ranging from 2 to 6 mm in diameter with a hypodermic needle. A group of collected oocytes was incubated in maturation medium CRML 1066 to obtain metaphase II oocytes. Succinate, lactate and glucose-6-phosphate dehydrogenase activities in just collected meiotically immature and in vitro matured oocytes were assayed cytochemically. Microdensitometric measurements were made with a Vickers M85a scanning microdensitometer. Our findings show that: 1) succinate dehydrogenase activity was significantly increased in matured oocytes; 2) lactate dehydrogenase activity was present and very strong in immature oocytes but was detectable in only about 50 % of matured oocytes, with significantly lower integrated optical density values; 3) glucose-6-phosphate dehydrogenase activity was very high in immature oocytes but significantly decreased after in vitro maturation; 4) there was no linear correlation between the integrated optical densities of the three enzymatic activities and the diameters of the oocytes. We suggest that the ability to utilize glucose may appear earlier in bovine oocytes than in other species and takes place at the time of maturation.
Key words: Preovulatory oocytes - cattle - quantitative cytochemistry - succinate dehydrogenase - lactate dehydrogenase - glucose-6-phosphate dehydrogenase
Introduction In all animals, oocytes that become arrested at prophase I during the growth period resume meiosis near or at the end of their growth. The process in which prophase I-arrested oocytes resume meiosis is called maturation (Masui, 1985). Cell metabolism during oocyte growth is certainly different from that during somatic cell growth. It appears that the special cell metabolism in growing oocytes plays a role in Correspondence to: B. Ferrandi
90
B. Ferrandi et al.
stabilizing the arrested meiosis. It may be the cessation of this special metabolism at the end of the growth period that destabilizes the arrested meiosis (Masui, 1985). Most of our information about levels of metabolic enzymes and metabolites of the mammalian ovum comes from studies of mouse and rabbit ova (Wolf, 1982). There are almost no data for ova of mammalian domestic animals. Therefore we were interested to investigate the metabolic activity of the bovine oocyte during growth and maturation especially in view of the technique of in vitro fertilization and implantation. Since mammalian oocytes acquire the ability to resume meiotic maturation spontaneously when removed from the ovary and cultured in vitro, we employed this technique to study some aspects of the metabolism of these cells. In vitro fertilization techniques in domestic animals, especially in cattle, are mainly based on the possibility of successful in vitro culturing of meiotic-arrested oocytes to obtain metaphase II oocytes competent to undergo fertilization. If similar systems could be established for human oocyte development it might become possible to replace the hyperstimulatory techniques currently in use in in vitro fertilization clinics. Since there are many data indicating that metabolism in mammalian oocytes is different from that in more differentiated cells (De Schepper et al., 1985), we used cytochemical quantitative assays to measure succinate dehydrogenase, lactate dehydrogenase, and glucose6-phosphate dehydrogenase activities in preovulatory oocytes and in oocytes matured in vitro collected from ovaries of non-pregnant cows. Virtually all studies of enzyme activities in oocytes (and preimplantation embryos) have been performed with biochemical methods, which require homogenation, which has several obvious disadvantages, e.g., disruption of the cells which gives average data per cell and no information about the localization of enzyme activity. Therefore, determination of enzyme activities in single cells with cytochemical staining methods should overcome these problems. Especially in studies of such unique cells as oocytes and with respect to fertilization and embryonic development, investigations of single cells may be of utmost importance, and in addition, the enzyme activity of each individual cell can be correlated with other cytological parameters such as, for exemple, cell diameter.
Materials and Methods Sampling: Growing oocytes, mostly at the genninal vesicle stage, as detected for some fixed and Giemsastained (Sigma Aldrich, Milano) oocytes immediately after recovery, were obtained by puncturing follicles ranging from 2 to 6 mm in diameter from 60 ovaries of 30 non-pregnant cows with a hypodennic needle. The oocytes were collected in a drop of phosphate buffer, pH 7.4, containing 0.1 % polyvinyl pyrrolidone (Sigma Aldrich, Milano) and only oocytes with compact cumulus, regular-shaped cytoplasm and intact germinal vesicle were used. Preovulatory oocytes were divided into two groups: the first served to study oocytes in the growth phase, the second for in vitro maturation. The oocytes of the first group were completely freed from any adherent granulosa cells immediately after recovery by a fme glass micropipette. Some of these oocytes were incorporated into a thin film of polyacrylamide gel by the method of van Noorden et al. (1982); the others were kept in suspension. Immature oocytes trapped in the transparent film and the suspended cells were then processed for cytochemical reactions. In vitro maturation: The oocytes for the in vitro maturation group were incubated with the cumulus intact for 24 h at 39°C in 5 % CO2 in the maturation medium CRML 1066 (Sigma Aldrich, Milano) supplemented with 10% fetal calf serum (Sigma Aldrich, Milano) and 1 % antibiotic/antimycotic solution (Sigma Aldrich, Milano) to obtain oocytes at the metaphase IT stage. At the end of incubation, the matured oocytes were decumulated, one group were incorporated into a thin film of polyacrylamide gel, another group kept in suspension and then both analysed enzyme histochemically. Cytochemical reactions: Meiotically immature and in vitro-matured oocytes, both incorporated in a thin film of polyacrylamide gel and in suspension, were processed for cytochemical detection of succinate dehydrogenase (SDH, E.C. 1.3.99.1) activity (according to van Noorden et al., 1983a), lactate dehydrogenase
Enzymatic activities in bovine oocytes
91
Table 1. SDH, LDH and G6PDH activities of bovine immature oocytes and oocytes matured in vitro. hnmature oocytes SDH
LDH
G6PDH
100
100
100
128.47 ± 18.47 (14.37) 153.79±31.18 (20.27) Oocyte diameter (1J.Ill) 151.59± 14.06 (9.28)
120.72 ± 22.52 (18.65) 150.39 ± 26.60 (17.78) 150.69 ± 8.07 (5.36)
109.75 ± 13.89 (12.65) 117.28 ± 16.84 (14.35) 153.88±7.74 (5.03)
Oocytes matured in vitro SDH
LDH
G6PDH
100*
100
72.77± 10.50 (14.42) 91.66± 13.04 (14.22) 146.02 ± 8.22 (5.63)
65.07 ± 12.73 (19.65) 84.06 ± 22.12 (26.31) 147.92±8.21 (5.55)
Number of observations Integrated optical density Integrated area
Number of observations Integrated optical density Integrated area
100
143.21 ± 28.57 (19.94) 180.19 ± 41.21 (22.87) Oocyte diameter (1J.Ill) 152.42±7.57 (4.97)
The values are mean ± S.D. Coefficient of variation in parentheses. *LDH activity was detectable in only 46 matured oocytes.
(LDH, E.C. 1.1.1.27) and glucose-6-phosphate dehydrogenase (G6PDH, E.C. 1.1.1.49) activities (according to De Schepper et al., 1985). Three control media were employed: one without the specific substrates, succinate (Sigma Aldrich, Milano), lactate (Serva, Heidelberg) or glucose-6-phosphate (Sigma Aldrich, Milano); another without the coenzymes NAD (Sigma Aldrich, Milano) or NADP (Sigma Aldrich, Milano) for LDH or G6PDH and the third without either substrate and coenzyme, to measure non-specific dehydrogenase activities. In all three cytochemical reactions, tetranitro BT (TNBT, Sigma Aldrich, Milano) was the final electron acceptor.
Determination of enzymatic activities: Microphotometric measurements (integrated optical density, 1. O. D.), were carried out on 100 bovine oocytes for each type of cell. Cytochemical determinations were made with a Vickers M85a scanning microdensitometer at 534 om wavelength, which is the absorption maximum for precipitated TNBT formazan (van Noorden and Tas, 1980; van Noorden and Tas, 1981; van Noorden et al., 1983b). In addition, the relationship between the increase in integrated absorbance at 534 om and the duration of incubation was linear, which is a prerequisite for cytochemical enzyme investigation (Kugler, 1981; van Noorden et al., 1983b). The microdensitometer had a lOx objective, a lOx eyepiece, a dry condenser and a 2 Itm diameter flying spot; the scan time for SDH activity was 4 sec and for LDH and G6PDH 2 sec; the band width was 40 om, the threshold 0.3 and the mask diameter 160 Itm. Integrated optical density and integrated area were expressed in arbitrary units. Some single oocytes were examined with a Carl Zeiss Universal Microspectrophotometer System to obtain three-dimensional representations of the absorbance profiles along the scanning lines of the X-axis (ISO-plot). The diameters of all cells were also measured. Statistical analysis: The data were analyzed statistically by analysis of variance with the Scheffe test for multiple comparison. Only differences of p :s; 0.01 were regarded as statistically significant.
92
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Figs. 1-6. Integrated optical density values (I. O. D.) in arbitrary units (a. u.) versus oocyte diameter (0) and versus integrated area (_). Integrated area, expressed in arbitrary units (a. u.) is the area within the cytoplasm occupied by the formazan precipitate due to enzyme activities.
Results The oocyte morphology was well preserved for both the cells incorporated in the matrix of a thin film of transparent polyacrylamide prior to incubation in the cytochemical media and in those processed in suspension. The precipitation of formazan due to enzyme activities occurred entirely within the cell cytoplasm, leaving the zona pellucida virtually unstained; no formazan was found in the incubation medium or precipitated in the polyacrylamide matrix outside the cells, which means there was no leakage of enzyme molecules, reduced intermediates or formazan out the cell. The microdensitometric measurements of individual oocytes trapped in a polyacrylamide matrix or processed in suspension showed no statistically significant differences, and thus our data are discussed without reference to the different methods employed before cytochemical staining. In oocytes incubated in the cytochemical control media, no formazan production was detected. There was, therefore, no "nothing dehydrogenase" activity. SDH activity was significantly increased in bovine oocytes after in vitro maturation (Table 1). LDH activity was present and very strong in immature oocytes but was cytochemically detectable in only 46 % of matured oocytes with significantly lower I.O.D. values (Table 1). G6PDH activity was very high in immature oocytes but decreased significantly after in vitro maturation (Table 1). Furthermore, there was a fair correlation between the LO.D. values and the integrated area for the three enzymatic activities (area within the cytoplasm occupied by the formazan
94
B. Ferrandi et al.
Figs. 7-8. Absorbance profile along the scanning lines of the X-axis of two immature oocytes processed for quantitative cytochemical detection of LDU activity (Fig. 7: diameter of oocyte 1801!m; Fig. 8: diameter of oocyte 172l!m).
precipitate due to enzyme activities) (Figs. 1-6). On the contrary, no such correlation was observed between I.O.D. values and oocyte diameter (Figs. 1-6). The lack of positive correlation between the oocyte diameter and the intensity of the reaction is emphasized in the three-dimensional representation of the absorbance profiles (Figs. 7-8).
Discussion The mammalian ovum represents a metabolically inert cell able to fulfill a variety of biosynthetic functions in response to the activating stimulus provided by capacitated sperm penetration or exposure to parthenogenetic agents (Wolf, 1982). However, some enzymatic activities, such as those involved in energy metabolism or synthesis and accumulation of specific proteins, have been demonstrated in growing oocytes (for review see Zaneveld and
Enzymatic activities in bovine oocytes
95
Chatterton, 1982; Metz and Monroy, 1985). In some species, oocytes acquire the ability to resume meiotic maturation spontaneously when removed from the ovary or cultured in vitro. Generally the full grown oocyte is already metabolically fairly active and the induction of maturation may result in metabolic changes. Our quantitative cytochemical data for some enzymatic activities of bovine preovulatory oocytes examined before and after in vitro maturation lead to interesting considerations. The significant increase in SDH activity in mature oocytes supports the idea that there is an increase in the absolute number of mitochondria per oocyte, as hypothesized for the mouse oocyte by Vivarelli et al. (1976) rather than increased enzyme activity per mitochondrion since oocyte mitochondrial configuration does not change during oogenesis (for review see Szollosi, 1972). LDH activity was extremely high in preovulatory oocytes, as in mouse preovulatory female germinal cells (De Schepper et al., 1985), and this leads us to hypothesize that this high enzyme level may be related, as in rodent oocytes, to the utilization of lactate and pyruvate as energy sources during oocyte growth (Mangia et al., 1976). Furthermore, we believe that the increase in LDH activity during oocytes growth also results from actual synthesis of the enzyme protein itself, as demonstrated in mouse oocytes by direct biochemical assays (Mangia et al., 1976). However, in mouse oocytes LDH activity remains constant during ovulation and first two days of postfertilization embryonic development (Mangia et al., 1976), while in bovine oocytes we found a dramatic cessation of LDH activity in about 50% of the matured oocytes, with the remaining 50% showing significantly lower I.O.D. values. It seems thus reasonable to assume that the ability to utilize glucose, which does not develop in the mouse until after the onset of cleavage (Biggers et al., 1966), could take place earlier in bovine oocytes, i.e. at the time of maturation. On the other hand, as observed in the mouse after the second embryonic day, the decrease or cessation of such activity may not be due to enzyme inhibition but may result from the diseappearance of the enzyme protein itself (Spielman et al., 1974a, b). G6PDH, the key-enzyme in the penthose phosphate pathway was very active in bovine preovulatory oocytes, as demonstrated in the mouse biochemically (Brinster, 1976; Mangia and Epstein, 1975) and cytochemically (De Schepper et al., 1985). The reason for this high activity in oocytes is uncertain. The production of NADPH is e.g. important for synthetic processes, particularly lipid synthesis. An other function of G6PDH is to form ribose and deoxyribose for the RNA and DNA synthesis. The decreasing G6PDH activity during oocyte maturation may be correlated with partial enzyme degradation, as hypothesized for the mouse during the third and fourth days of development (Mangia et al., 1976). Finally, the lack of any correlation between the I.O.D. values of the three enzymatic activities and oocyte diameters in our study partially disagrees with the results of Mangia and Epstein (1975) for G6PDH and LDH activities in mouse oocytes, in which both enzymatic activities increased with oocyte volume, at least up to a determined size. We consider the measured diameter proportional to oocyte volume, assuming the female gamete to be a sphere. Our findings are of interest in the context of comparative studies of different species with different evolution, furthermore our data show that understanding aspects of oocyte metabolism are important for techniques used in in vitro maturation and in in vitro fertilization.
References Biggers JD, Wittingham 00, and Donahue RP (1967) The pattem of energy metabolism in the mouse oocyte and zygote. Proc Nat Acad Sci USA 58: 560-567. Brinster RL (1966) Glucose-6-phosphate dehydrogenase activity in the preimplantation mouse embryo. Biochem J 101: 161-163.
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De Schepper GG, van Noorden CJF, and Koperdraad F (1985) A cytochemical method for measuring enzyme activity in individual preovulatory mouse oocytes. J Reprod Fert 74: 709-716. Kugler P (1981) Kinetic and morphometric measurements of enzyme reactions in tissue sections with a new instrumental setup. Histochemistry 71: 433-439. Mangia F, and Epstein CJ (1975) Biochemical studies of growing mouse oocytes: preparation of oocyte and analysis of glucose-6-phosphate dehydrogenase and lactate dehydrogenase activities. Develop Bioi 45: 211-220. Mangia F, Erickson RP, and Epstein CJ (1976) Synthesis of LDH-I during mammalian oogenesis and early development. Develop Bioi 54: 146-150. Masui Y (1985) Meiotic arrest in animal oocytes. In: Metz CB, Monroy A (Eds) Biology of fertilization. Academic Press, Orlando, San Diego, New York, London, Toronto, Montreal, Sydney, Tokyo, pp 189-219. Metz CB, and Monroy A (1985) Biology of fertilization. Academic Press, Orlando, San Diego, New York, London, Toronto, Montreal, Sydney, Tokyo. Spielman H, Erickson RP, and Epstein CJ (1974a) Immunochemical studies of lactate dehydrogenase and glucose-6-phosphate dehydrogenase in preimplantation mouse embryos. J Reprod Fert 40: 367-373. Spielman H, Erickson RP, and Epstein CJ (1974b) The production of antibodies against mammalian LDH-1. Anal Biochem 59: 462-467. Szollosi D (1972) Changes of some organelles during oogenesis in mammals. In: Biggers JD, Schuetz AW (Eds) Oogenesis. University Park Press, Baltimore, pp 77-84. (cited by Vivarelli et al., 1976). van Noorden CJF, Bhattacharya RD, and Vogels IMC (1983 a) Enzyme cytochemical staining of individual cells with the use of a polyacrylamide carrier. Acta histochem 73: 71-78. van Noorden CJF, and Tas J (1980) Quantitative aspects of the cytochemical demonstration of glucose-6phosphate dehydrogenase with tetranitro BT studied in a model system of polyacrylamide films. Histochem J 12: 669-685. van Noorden CJF, and Tas J (1981) Model film studies in enzyme histochemistry with special reference to glucose-6-phosphate dehydrogenase. Histochem J 13: 187-206. van Noorden CJF, Tas J, and Vogel IMC (1983b) Cytophotometry of glucose-6-phosphate dehydrogenase activity in individual cells. Histochem J 15: 583-599. van Noorden CJF, Tas J, Vogels IMC, and De Schepper GG (1982) A new method for the enzyme cytochemical staining of individual cells with the use of a polyacrylamide carrier. Histochemistry 74: 171-181. Vivarelli E, Siracusa G, and Mangia F (1976) A histochemical study of succinate dehydrogenase in mouse oocytes and early embryos. J Reprod Fert 47: 149-150. Wolf DP (1982) The ovum before and afterfertilization. In: Zaneveld LDJ, Chatterton RT (Eds) Biochemistry of mammalian reproduction. John Wiley & Sons, New York, Chichester, Brisbane, Toronto, Singapore, pp 231-259. Zaneveld UD, and Chatterton RT (1982) Biochemistry of mammalian reproduction. John Wiley & Sons, New York, Chichester, Brisbane, Toronto, Singapore.
Ada
his'o~helRiea
Quantitative cytochemical study of some enzymatic activities in preovulatory bovine oocytes after in vitro maturation Bruno FerrandP), Fausto Cremonesti2), Renato Geiger2), Anna Lange Consiglio l ), Antino CarnevalP), and Franca PorcellP) I)Institute of Anatomy of Domestic Animals and 2)Institute of Veterinary Gynecology and Clinical Obstetrics, University of Milano, Via Celoria 10, 20133 Milano, Italy Accepted 1 June 1993
Summary In the present study quantitative cytochemical assays were used to measure some enzymatic activities in situ in bovine meiotically immature oocytes and oocytes matured in vitro, since the special metabolic activity of the growing oocytes may be a pivotal factor in stabilizing the meiotically arrested oocytes. Modifications of this particular metabolism might destabilize the arrested meiosis. Preovulatory oocytes, mostly at the germinal vesicle stage, were obtained by puncturing follicles ranging from 2 to 6 mm in diameter with a hypodermic needle. A group of collected oocytes was incubated in maturation medium CRML 1066 to obtain metaphase II oocytes. Succinate, lactate and glucose-6-phosphate dehydrogenase activities in just collected meiotically immature and in vitro matured oocytes were assayed cytochemically. Microdensitometric measurements were made with a Vickers M85a scanning microdensitometer. Our findings show that: 1) succinate dehydrogenase activity was significantly increased in matured oocytes; 2) lactate dehydrogenase activity was present and very strong in immature oocytes but was detectable in only about 50 % of matured oocytes, with significantly lower integrated optical density values; 3) glucose-6-phosphate dehydrogenase activity was very high in immature oocytes but significantly decreased after in vitro maturation; 4) there was no linear correlation between the integrated optical densities of the three enzymatic activities and the diameters of the oocytes. We suggest that the ability to utilize glucose may appear earlier in bovine oocytes than in other species and takes place at the time of maturation.
Key words: Preovulatory oocytes - cattle - quantitative cytochemistry - succinate dehydrogenase - lactate dehydrogenase - glucose-6-phosphate dehydrogenase
Introduction In all animals, oocytes that become arrested at prophase I during the growth period resume meiosis near or at the end of their growth. The process in which prophase I-arrested oocytes resume meiosis is called maturation (Masui, 1985). Cell metabolism during oocyte growth is certainly different from that during somatic cell growth. It appears that the special cell metabolism in growing oocytes plays a role in Correspondence to: B. Ferrandi
90
B. Ferrandi et al.
stabilizing the arrested meiosis. It may be the cessation of this special metabolism at the end of the growth period that destabilizes the arrested meiosis (Masui, 1985). Most of our information about levels of metabolic enzymes and metabolites of the mammalian ovum comes from studies of mouse and rabbit ova (Wolf, 1982). There are almost no data for ova of mammalian domestic animals. Therefore we were interested to investigate the metabolic activity of the bovine oocyte during growth and maturation especially in view of the technique of in vitro fertilization and implantation. Since mammalian oocytes acquire the ability to resume meiotic maturation spontaneously when removed from the ovary and cultured in vitro, we employed this technique to study some aspects of the metabolism of these cells. In vitro fertilization techniques in domestic animals, especially in cattle, are mainly based on the possibility of successful in vitro culturing of meiotic-arrested oocytes to obtain metaphase II oocytes competent to undergo fertilization. If similar systems could be established for human oocyte development it might become possible to replace the hyperstimulatory techniques currently in use in in vitro fertilization clinics. Since there are many data indicating that metabolism in mammalian oocytes is different from that in more differentiated cells (De Schepper et al., 1985), we used cytochemical quantitative assays to measure succinate dehydrogenase, lactate dehydrogenase, and glucose6-phosphate dehydrogenase activities in preovulatory oocytes and in oocytes matured in vitro collected from ovaries of non-pregnant cows. Virtually all studies of enzyme activities in oocytes (and preimplantation embryos) have been performed with biochemical methods, which require homogenation, which has several obvious disadvantages, e.g., disruption of the cells which gives average data per cell and no information about the localization of enzyme activity. Therefore, determination of enzyme activities in single cells with cytochemical staining methods should overcome these problems. Especially in studies of such unique cells as oocytes and with respect to fertilization and embryonic development, investigations of single cells may be of utmost importance, and in addition, the enzyme activity of each individual cell can be correlated with other cytological parameters such as, for exemple, cell diameter.
Materials and Methods Sampling: Growing oocytes, mostly at the genninal vesicle stage, as detected for some fixed and Giemsastained (Sigma Aldrich, Milano) oocytes immediately after recovery, were obtained by puncturing follicles ranging from 2 to 6 mm in diameter from 60 ovaries of 30 non-pregnant cows with a hypodennic needle. The oocytes were collected in a drop of phosphate buffer, pH 7.4, containing 0.1 % polyvinyl pyrrolidone (Sigma Aldrich, Milano) and only oocytes with compact cumulus, regular-shaped cytoplasm and intact germinal vesicle were used. Preovulatory oocytes were divided into two groups: the first served to study oocytes in the growth phase, the second for in vitro maturation. The oocytes of the first group were completely freed from any adherent granulosa cells immediately after recovery by a fme glass micropipette. Some of these oocytes were incorporated into a thin film of polyacrylamide gel by the method of van Noorden et al. (1982); the others were kept in suspension. Immature oocytes trapped in the transparent film and the suspended cells were then processed for cytochemical reactions. In vitro maturation: The oocytes for the in vitro maturation group were incubated with the cumulus intact for 24 h at 39°C in 5 % CO2 in the maturation medium CRML 1066 (Sigma Aldrich, Milano) supplemented with 10% fetal calf serum (Sigma Aldrich, Milano) and 1 % antibiotic/antimycotic solution (Sigma Aldrich, Milano) to obtain oocytes at the metaphase IT stage. At the end of incubation, the matured oocytes were decumulated, one group were incorporated into a thin film of polyacrylamide gel, another group kept in suspension and then both analysed enzyme histochemically. Cytochemical reactions: Meiotically immature and in vitro-matured oocytes, both incorporated in a thin film of polyacrylamide gel and in suspension, were processed for cytochemical detection of succinate dehydrogenase (SDH, E.C. 1.3.99.1) activity (according to van Noorden et al., 1983a), lactate dehydrogenase
Enzymatic activities in bovine oocytes
91
Table 1. SDH, LDH and G6PDH activities of bovine immature oocytes and oocytes matured in vitro. hnmature oocytes SDH
LDH
G6PDH
100
100
100
128.47 ± 18.47 (14.37) 153.79±31.18 (20.27) Oocyte diameter (1J.Ill) 151.59± 14.06 (9.28)
120.72 ± 22.52 (18.65) 150.39 ± 26.60 (17.78) 150.69 ± 8.07 (5.36)
109.75 ± 13.89 (12.65) 117.28 ± 16.84 (14.35) 153.88±7.74 (5.03)
Oocytes matured in vitro SDH
LDH
G6PDH
100*
100
72.77± 10.50 (14.42) 91.66± 13.04 (14.22) 146.02 ± 8.22 (5.63)
65.07 ± 12.73 (19.65) 84.06 ± 22.12 (26.31) 147.92±8.21 (5.55)
Number of observations Integrated optical density Integrated area
Number of observations Integrated optical density Integrated area
100
143.21 ± 28.57 (19.94) 180.19 ± 41.21 (22.87) Oocyte diameter (1J.Ill) 152.42±7.57 (4.97)
The values are mean ± S.D. Coefficient of variation in parentheses. *LDH activity was detectable in only 46 matured oocytes.
(LDH, E.C. 1.1.1.27) and glucose-6-phosphate dehydrogenase (G6PDH, E.C. 1.1.1.49) activities (according to De Schepper et al., 1985). Three control media were employed: one without the specific substrates, succinate (Sigma Aldrich, Milano), lactate (Serva, Heidelberg) or glucose-6-phosphate (Sigma Aldrich, Milano); another without the coenzymes NAD (Sigma Aldrich, Milano) or NADP (Sigma Aldrich, Milano) for LDH or G6PDH and the third without either substrate and coenzyme, to measure non-specific dehydrogenase activities. In all three cytochemical reactions, tetranitro BT (TNBT, Sigma Aldrich, Milano) was the final electron acceptor.
Determination of enzymatic activities: Microphotometric measurements (integrated optical density, 1. O. D.), were carried out on 100 bovine oocytes for each type of cell. Cytochemical determinations were made with a Vickers M85a scanning microdensitometer at 534 om wavelength, which is the absorption maximum for precipitated TNBT formazan (van Noorden and Tas, 1980; van Noorden and Tas, 1981; van Noorden et al., 1983b). In addition, the relationship between the increase in integrated absorbance at 534 om and the duration of incubation was linear, which is a prerequisite for cytochemical enzyme investigation (Kugler, 1981; van Noorden et al., 1983b). The microdensitometer had a lOx objective, a lOx eyepiece, a dry condenser and a 2 Itm diameter flying spot; the scan time for SDH activity was 4 sec and for LDH and G6PDH 2 sec; the band width was 40 om, the threshold 0.3 and the mask diameter 160 Itm. Integrated optical density and integrated area were expressed in arbitrary units. Some single oocytes were examined with a Carl Zeiss Universal Microspectrophotometer System to obtain three-dimensional representations of the absorbance profiles along the scanning lines of the X-axis (ISO-plot). The diameters of all cells were also measured. Statistical analysis: The data were analyzed statistically by analysis of variance with the Scheffe test for multiple comparison. Only differences of p :s; 0.01 were regarded as statistically significant.
92
B. Ferrandi et al. SUCCINATE DEHYDROGENASE IMMATURE OOCYTES
SUCCINATE DEHYDROGENASE MATURE OOCYTES
450 ,----------, 200
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Figs. 1-6. Integrated optical density values (I. O. D.) in arbitrary units (a. u.) versus oocyte diameter (0) and versus integrated area (_). Integrated area, expressed in arbitrary units (a. u.) is the area within the cytoplasm occupied by the formazan precipitate due to enzyme activities.
Results The oocyte morphology was well preserved for both the cells incorporated in the matrix of a thin film of transparent polyacrylamide prior to incubation in the cytochemical media and in those processed in suspension. The precipitation of formazan due to enzyme activities occurred entirely within the cell cytoplasm, leaving the zona pellucida virtually unstained; no formazan was found in the incubation medium or precipitated in the polyacrylamide matrix outside the cells, which means there was no leakage of enzyme molecules, reduced intermediates or formazan out the cell. The microdensitometric measurements of individual oocytes trapped in a polyacrylamide matrix or processed in suspension showed no statistically significant differences, and thus our data are discussed without reference to the different methods employed before cytochemical staining. In oocytes incubated in the cytochemical control media, no formazan production was detected. There was, therefore, no "nothing dehydrogenase" activity. SDH activity was significantly increased in bovine oocytes after in vitro maturation (Table 1). LDH activity was present and very strong in immature oocytes but was cytochemically detectable in only 46 % of matured oocytes with significantly lower I.O.D. values (Table 1). G6PDH activity was very high in immature oocytes but decreased significantly after in vitro maturation (Table 1). Furthermore, there was a fair correlation between the LO.D. values and the integrated area for the three enzymatic activities (area within the cytoplasm occupied by the formazan
94
B. Ferrandi et al.
Figs. 7-8. Absorbance profile along the scanning lines of the X-axis of two immature oocytes processed for quantitative cytochemical detection of LDU activity (Fig. 7: diameter of oocyte 1801!m; Fig. 8: diameter of oocyte 172l!m).
precipitate due to enzyme activities) (Figs. 1-6). On the contrary, no such correlation was observed between I.O.D. values and oocyte diameter (Figs. 1-6). The lack of positive correlation between the oocyte diameter and the intensity of the reaction is emphasized in the three-dimensional representation of the absorbance profiles (Figs. 7-8).
Discussion The mammalian ovum represents a metabolically inert cell able to fulfill a variety of biosynthetic functions in response to the activating stimulus provided by capacitated sperm penetration or exposure to parthenogenetic agents (Wolf, 1982). However, some enzymatic activities, such as those involved in energy metabolism or synthesis and accumulation of specific proteins, have been demonstrated in growing oocytes (for review see Zaneveld and
Enzymatic activities in bovine oocytes
95
Chatterton, 1982; Metz and Monroy, 1985). In some species, oocytes acquire the ability to resume meiotic maturation spontaneously when removed from the ovary or cultured in vitro. Generally the full grown oocyte is already metabolically fairly active and the induction of maturation may result in metabolic changes. Our quantitative cytochemical data for some enzymatic activities of bovine preovulatory oocytes examined before and after in vitro maturation lead to interesting considerations. The significant increase in SDH activity in mature oocytes supports the idea that there is an increase in the absolute number of mitochondria per oocyte, as hypothesized for the mouse oocyte by Vivarelli et al. (1976) rather than increased enzyme activity per mitochondrion since oocyte mitochondrial configuration does not change during oogenesis (for review see Szollosi, 1972). LDH activity was extremely high in preovulatory oocytes, as in mouse preovulatory female germinal cells (De Schepper et al., 1985), and this leads us to hypothesize that this high enzyme level may be related, as in rodent oocytes, to the utilization of lactate and pyruvate as energy sources during oocyte growth (Mangia et al., 1976). Furthermore, we believe that the increase in LDH activity during oocytes growth also results from actual synthesis of the enzyme protein itself, as demonstrated in mouse oocytes by direct biochemical assays (Mangia et al., 1976). However, in mouse oocytes LDH activity remains constant during ovulation and first two days of postfertilization embryonic development (Mangia et al., 1976), while in bovine oocytes we found a dramatic cessation of LDH activity in about 50% of the matured oocytes, with the remaining 50% showing significantly lower I.O.D. values. It seems thus reasonable to assume that the ability to utilize glucose, which does not develop in the mouse until after the onset of cleavage (Biggers et al., 1966), could take place earlier in bovine oocytes, i.e. at the time of maturation. On the other hand, as observed in the mouse after the second embryonic day, the decrease or cessation of such activity may not be due to enzyme inhibition but may result from the diseappearance of the enzyme protein itself (Spielman et al., 1974a, b). G6PDH, the key-enzyme in the penthose phosphate pathway was very active in bovine preovulatory oocytes, as demonstrated in the mouse biochemically (Brinster, 1976; Mangia and Epstein, 1975) and cytochemically (De Schepper et al., 1985). The reason for this high activity in oocytes is uncertain. The production of NADPH is e.g. important for synthetic processes, particularly lipid synthesis. An other function of G6PDH is to form ribose and deoxyribose for the RNA and DNA synthesis. The decreasing G6PDH activity during oocyte maturation may be correlated with partial enzyme degradation, as hypothesized for the mouse during the third and fourth days of development (Mangia et al., 1976). Finally, the lack of any correlation between the I.O.D. values of the three enzymatic activities and oocyte diameters in our study partially disagrees with the results of Mangia and Epstein (1975) for G6PDH and LDH activities in mouse oocytes, in which both enzymatic activities increased with oocyte volume, at least up to a determined size. We consider the measured diameter proportional to oocyte volume, assuming the female gamete to be a sphere. Our findings are of interest in the context of comparative studies of different species with different evolution, furthermore our data show that understanding aspects of oocyte metabolism are important for techniques used in in vitro maturation and in in vitro fertilization.
References Biggers JD, Wittingham 00, and Donahue RP (1967) The pattem of energy metabolism in the mouse oocyte and zygote. Proc Nat Acad Sci USA 58: 560-567. Brinster RL (1966) Glucose-6-phosphate dehydrogenase activity in the preimplantation mouse embryo. Biochem J 101: 161-163.
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De Schepper GG, van Noorden CJF, and Koperdraad F (1985) A cytochemical method for measuring enzyme activity in individual preovulatory mouse oocytes. J Reprod Fert 74: 709-716. Kugler P (1981) Kinetic and morphometric measurements of enzyme reactions in tissue sections with a new instrumental setup. Histochemistry 71: 433-439. Mangia F, and Epstein CJ (1975) Biochemical studies of growing mouse oocytes: preparation of oocyte and analysis of glucose-6-phosphate dehydrogenase and lactate dehydrogenase activities. Develop Bioi 45: 211-220. Mangia F, Erickson RP, and Epstein CJ (1976) Synthesis of LDH-I during mammalian oogenesis and early development. Develop Bioi 54: 146-150. Masui Y (1985) Meiotic arrest in animal oocytes. In: Metz CB, Monroy A (Eds) Biology of fertilization. Academic Press, Orlando, San Diego, New York, London, Toronto, Montreal, Sydney, Tokyo, pp 189-219. Metz CB, and Monroy A (1985) Biology of fertilization. Academic Press, Orlando, San Diego, New York, London, Toronto, Montreal, Sydney, Tokyo. Spielman H, Erickson RP, and Epstein CJ (1974a) Immunochemical studies of lactate dehydrogenase and glucose-6-phosphate dehydrogenase in preimplantation mouse embryos. J Reprod Fert 40: 367-373. Spielman H, Erickson RP, and Epstein CJ (1974b) The production of antibodies against mammalian LDH-1. Anal Biochem 59: 462-467. Szollosi D (1972) Changes of some organelles during oogenesis in mammals. In: Biggers JD, Schuetz AW (Eds) Oogenesis. University Park Press, Baltimore, pp 77-84. (cited by Vivarelli et al., 1976). van Noorden CJF, Bhattacharya RD, and Vogels IMC (1983 a) Enzyme cytochemical staining of individual cells with the use of a polyacrylamide carrier. Acta histochem 73: 71-78. van Noorden CJF, and Tas J (1980) Quantitative aspects of the cytochemical demonstration of glucose-6phosphate dehydrogenase with tetranitro BT studied in a model system of polyacrylamide films. Histochem J 12: 669-685. van Noorden CJF, and Tas J (1981) Model film studies in enzyme histochemistry with special reference to glucose-6-phosphate dehydrogenase. Histochem J 13: 187-206. van Noorden CJF, Tas J, and Vogel IMC (1983b) Cytophotometry of glucose-6-phosphate dehydrogenase activity in individual cells. Histochem J 15: 583-599. van Noorden CJF, Tas J, Vogels IMC, and De Schepper GG (1982) A new method for the enzyme cytochemical staining of individual cells with the use of a polyacrylamide carrier. Histochemistry 74: 171-181. Vivarelli E, Siracusa G, and Mangia F (1976) A histochemical study of succinate dehydrogenase in mouse oocytes and early embryos. J Reprod Fert 47: 149-150. Wolf DP (1982) The ovum before and afterfertilization. In: Zaneveld LDJ, Chatterton RT (Eds) Biochemistry of mammalian reproduction. John Wiley & Sons, New York, Chichester, Brisbane, Toronto, Singapore, pp 231-259. Zaneveld UD, and Chatterton RT (1982) Biochemistry of mammalian reproduction. John Wiley & Sons, New York, Chichester, Brisbane, Toronto, Singapore.