MOLECULAR REPRODUCTION AND DEVELOPMENT 26222-226 (1990)
Analysis of Protein Synthesis in Morphologically Classified Bovine Follicular Oocytes Before and After Maturation In Vitro P.M.M. KASTROP,' M.M. BEVERS,' O.H.J. DESTRkE?
AND
TH.A.M. KRUIP'
'Department of Herd Health and Reproduction, Faculty of Veterinary Medicine, University of Utrecht, and 'Hubrecht Laboratory, The Netherlands Institute for Developmental Biology, Utrecht, The Netherlands
ABSTRACT Bovine cumulus oocyte complexes (COCs) were isolated from antral ovarian follicles (4-8 mm). Immature COCs were classified into four categories, based on the homogeneity and clearness of the ooplasm and the transparency and compactness of the cumulus investment. In this study, the incorporation of TCA-precipitable 35S-methionineand the protein synthesis patterns of oocytes of these four categories were examined. Before maturation in vitro, similar incorporation rates and identical protein synthesis patterns were observed between oocytes of categories 1-3. Immature oocytes of category 4 showed reduced incorporation rates and exhibited aberrant protein synthesis patterns. After maturation in vitro, the patterns of category 4 oocytes were identical with the patterns of those in categories 1-3. The incorporation of 35S-methionine into in vitro matured oocytes was lower ( P < .001) in all categories. Based on these results, it is concluded that the initial classification of oocytes into four categories can be reduced to two categories.
follicles between 4 and 8 mm, into four categories. The classification is based on morphological features of both the ooplasm and the cumulus investment. Previously, the ultrastructural characteristics and the capacity to mature in vitro of the four categories were investigated (De Loos et al., 1989). Although the morphological differences between categories 1-4 oocytes were confirmed at the ultrastructural level, they were not reflected in different maturation capacities. Only the degenerating category 4 oocytes exhibited a decreased capacity to mature in vitro (De Loos et al., 1989). In this study, protein synthesis patterns of individual oocytes of the four categories were analysed, both before and after in vitro maturation, to establish whether the observed morphological features are reflected in biochemical differences.
MATERIALS AND METHODS Collection and Culture of Cumulus Oocyte Complexes Bovine ovaries, obtained a t a local slaughterhouse, were rinsed and collected in Dulbecco's phosphate bufKey Words: Cow, Classification, Cumulus oocyte fered saline a t 30°C. Precautions were taken to avoid complexes, In vitro maturation bacterial contamination. The ovaries were transported to the laboratory within 1 h after slaughter. Antral follicles between 4 and 8 mm were punctured, and the INTRODUCTION follicular fluid aspirates were pooled in a conical cenThe percentage of oocytes which mature properly in trifuge tube. The settled COCs were recovered from the vitro and achieve developmental competence has been follicular fluid after 15 min of sedimentation and subclaimed to be related to both the size of the follicles, sequently washed in Dulbecco's phosphate buffered safrom which oocytes were recovered, and the presence of line, supplemented with 4 mg/ml bovine serum albua cumulus investment (Sato et al., 1977; Leibfried and min, 0.36 mM pyruvate, 23.8 mM lactate, and 5.5 mM First, 1979; Fukui and Sakuma, 1980; Staigmiller and glucose (medium A). COCs were classified under a steMoor, 1984; Critser et al., 1986; Younis et al., 1989). In reomicroscope (magnification 50 x ) and divided into spite of the application of these selection criteria, the four categories, based on the homogeneity and clearyield of advanced bovine preimplantation embryos is still quite low (Lu et al., 1987; Xu et al., 1987; Goto et al., 1988; Fayrer-Hosken et al., 1988; Stubbings et al., 1988; Utsumi et al., 1988). To improve this yield, reliable classification criteria will be necessary. September 26, 1989; accepted January 29, 1990. As an initial step towards a reliable and non-inva- Received Address reprint requests to P.M.M.Kastrop, Department of Herd sive classification system, we have distinguished im- Health and Reproduction, Faculty of Veterinary Medicine, University mature cumulus oocyte complexes (COCs), from antral of Utrecht, Yalelaan 7, 3584 CL Utrecht, The Netherlands. ~
~
0 1990 WILEY-LISS, INC.
~
PROTEIN SYNTHESIS IN BOVINE OOCYTES ness of the ooplasm and the transparency, integrity, and compactness of the cumulus investment (De Loos et al., 1989):Categories 1and 2 included COCs with a compact and multilayered cumulus investment and homogeneous ooplasm, although category 2 COCs exhibited a darker zone a t the periphery of the oocytes. Category 3 COCs possessed less compact and darker multilayered cumulus investments, whereas those of category 4 COCs were dark and irregularly expanded. The ooplasms in these COCs appeared irregular and contained dark clusters. The whole selection and classification procedure was carried out a t approximately 30°C and within 2-3 h after slaughter. Oocytes were matured in vitro by culture in Medium 199 with Earle's salts (Flow Laboratories, U.K.) supplemented with 10% heat-inactivated foetal calf serum, 1 pg/ml bLH, 2 pg/ml bFSH, and 1 kg/ml Oestradiol17p. Culture of COCs was carried out at 39°C in a humidified atmosphere of 5% C02 in air.
223
TABLE 1. Incorporation of "S-Methionine Into Bovine Oocyte Proteins, Before and After In Vitro Maturation (IVM) for the Four Morphological Categories "'S-methionine incoruoration (cpm/oocyte/36) Before N M After IVM
Category 1 2 3 4
9,118k 702 (15) 9,980 % 862 (12) 8,296 5 1,129 (12) 6,050 5 508 (12)
Values are mean rentheses.
f
5,661 2 296 (15) 6,138 f 522 (15) 3,748 t 284 (13) 3,292 f 428 (10)
S.E.M.for the number of oocytes in pa-
after in vitro maturation for 21 h in all four categories. Furthermore, category 4 oocytes exhibited a reduced incorporation (P 5 ,001) compared with categories 1-3 oocytes. The incorporation of 35S-methionine into the cumulus investment was about 20-50 times higher than was the incorporation into the oocytes (results not Radiolabelling and Electrophoresis shown), indicating the importance of a complete reCOCs were either radiolabelled immediately after moval of all cumulus cells. The patterns of de novo protein synthesis in individcollection for 3 h or cultured for 21 h, to accomplish in vitro maturation, and subsequently radiolabelled for 3 ual oocytes within the four categories are presented in h. Labelling was performed a t 39°C in medium A, con- Figure 1. Immature oocytes in categories 1-3 showed (specific activity > no differences in the protein synthesis patterns, taining 1mCi/ml ~-[~~Slmethionine 1,000 Ci/mmol; Amersham, U.K.). After labelling, the whereas category 4 oocytes exhibited an aberrant patoocytes were denuded of cumulus cells, and only intact tern (panel A). All the oocytes were in the germinal oocytes were collected and washed in medium A. These vesicle (GV)stage after the labelling period. The prooocytes were lysed individually by adding 20 kl SDS tein synthesis patterns of in vitro matured oocytes sample buffer (Laemmli, 19701, followed by heating at (panel B) showed no differences, i.e., the polypeptide 100°C for 2 min. Aliquots of 2 ~1 were used to deter- pattern of the in vitro matured category 4 oocyte was mine the amount of TCA-precipitable counts. The re- identical with those of the in vitro matured oocytes in mainder of each sample was stored at -20°C until elec- categories 1-3. The oocytes shown in panel B had extruded the first polar body after the 21 h culture and 3 trophoresis. Radiolabelled proteins were separated and resolved h labelling period. This indicated that nuclear matuby sodium dodecyl sulphate polyacrylamide gel electro- ration had been completed. Protein patterns of the immature oocytes in categophoresis (SDS-PAGE) according to the procedure of Laemmli (1970), using 8-15% linear gradient slab gels ries 1-3 (panel A), as compared to the in vitro matured of 0.75 mm thickness. Equal amounts of TCA-precipi- oocytes (panel B), showed marked differences, indicattable counts were loaded onto a gel. After electrophore- ing major changes in protein synthesis during meiotic sis, the gels were treated with AmplifyTM(Amersham, maturation. Protein synthesis patterns of both immaU.K.)for 30 min and dried under vacuum at 80°C. Sub- ture and in vitro matured oocytes in categories 1-3 sequently, the gels were exposed to Kodak-XAR5 films were always identical within a particular category. Also, no differences were observed between the patat -40°C for about 3 weeks. Mixtures of proteins with known molecular weight terns of in vitro matured oocytes in category 4 (results between 14.2 and 205 kDa (Sigma Chemicals Co., not shown). The heterogeneity within immature USA) were run simultaneously as standards and visu- oocytes in category 4 is shown in Figure 2. The majority of the protein synthesis patterns of these immature alized after electrophoresis by Coomassie staining. category 4 oocytes contained characteristic bands RESULTS which were also present in the polypeptide patterns of The incorporation of radiolabelled methionine into in vitro matured oocytes (lanes 2-5), but sometimes oocyte proteins was determined by TCA-precipitation, the polypeptide pattern resembled the pattern of imwhich enabled the loading of equal amounts of radio- mature oocytes in categories 1-3 (lane 1). activity per sample on a gel. The average incorporation DISCUSSION data of the four categories, both before and after in vitro maturation, are presented in Table 1.Analysis of In this study, we have investigated whether morphovariance revealed a decreased incorporation (P < .001) logical differences between immature COCs could be
224
P.M.M. KASTROP ET AL.
Fig. 1. Protein synthesis patterns of immature (A) and in vitro within a 21 h culture period (see Materials and Methods).The most matured (B)bovine oocytes. Categories 1-4 COCs were radiolabelled prominent differences between the patterns of immature categories with "S-methionine for 3 h and individually prepared for SDS-PAGE 1-3 and in vitro matured oocytes of categories 1-4 (116,95,70,54,33, and fluorography. In vitro maturation of oocytes was accomplished 26,22,and 14 kD)are marked by arrows in panel B.
confirmed at the level of protein synthetic capacities. As radiolabelling of newly synthesized proteins is dependent on amino acid incorporation, these data supplied additional information. The lower incorporation
rate in category 4 oocytes might be caused by a reduced number of junctional complexes. Ultrastructural investigations showed that category 4 oocytes possess less frequently occurring and not oocyte-penetrating junc-
PROTEIN SYNTHESIS IN BOVINE OOCYTES
Fig. 2. Protein synthesis patterns of five immature category 4 oocytes. Whereas the less frequently occurring pattern in lane 1 resembles the patterns of immature categories 1-3 oocytes (Fig. 1, panel A), the patterns in lanes 2-5 exhibited characteristics which were also observed in the patterns of in vitro matured oocytes (Fig. 1, panel B). COCs were radiolabelled for 3 h with '"S-methionine and individually prepared for SDS-PAGE and fluorography (see Materials and Methods).
tional complexes (De Loos et al., 1989). In addition, a regression of junctional complexes during maturation has been reported (Cran et al., 1980; Kruip et al.,
225
19831, which might explain the reduced incorporation after in vitro maturation. The protein synthesis patterns of both immature and in vitro matured oocytes in categories 1-4 did not reflect the observed morphological differences between the four categories. The majority of immature oocytes in category 4 exhibited a protein synthesis pattern which deviated from those in immature categories 1-3. This supports recently reported ultrastructural observations (De Loos et al., 19891, which indicated that category 4 consists of a heterogeneous population of degenerating oocytes. The differences between immature oocytes and in vitro matured oocytes in categories 1-3 revealed changes in protein synthesis during bovine oocyte maturation. Whether these changes occur subsequent to germinal vesicle breakdown (GVBD),as described for a number of other mammalian species (mouse: Golbus and Stein, 1976; Schultz and Wassarman, 1977; rabbit: Van Blerkom and McGaughey, 1978; sheep: Moor et al., 19811, is now under study. The remarkable resemblance of the protein synthesis patterns of most of the immature category 4 oocytes to the patterns of in vitro matured oocytes suggests an uncoupling of the nuclear and cytoplasmic maturation processes. This is supported by the data of Sun and Moor (19881, showing that the extensive changes in protein synthesis subsequent to GVBD is not dependent on transcription or the mixing of the karyoplasm with the cytoplasm after nuclear breakdown. A similar resemblance between these two groups was observed a t the ultrastructural level, as morphological characteristics of maturing oocytes were recognized in category 4 oocytes (De Loos et al., 1989). The uncoupling of nuclear and cytoplasmic maturation might be induced by the disturbed intercellular contact between cumulus cells and immature oocytes in category 4. Although morphological differences between oocyte categories, seen with light microscopy, were confirmed a t the ultrastructural level (De Loos et al., 19891, this study demonstrated that only category 4 differs obviously from categories 1-3 in protein synthesis patterns of the oocytes. Although a few immature oocytes in category 4 have protein synthesis patterns resembling those of immature oocytes in categories 1-3, the applied classification system distinguishes category 4 oocytes as heterogeneous and degenerating oocytes. Since these oocytes completed meiosis I a t a lower frequency in vitro (De Loos et al., 19891, they should be avoided in in vitro maturation and fertilization studies. So, the use of COCs with a compact and multilayered cumulus investment, i.e., categories 1-3 COCs, improves the proportion of oocytes which mature properly in vitro and achieve developmental competence. This confirms previous findings of Younis et al. (19891 and implies that based on these results categories 1-3 COCs may be pooled in practice for in vitro maturation and fertilization studies.
226
P.M.M. KASTROP ET AL.
ACKNOWLEDGMENTS This study was supported by grants of the Dutch Programme Committee for Agriculture Biotechnology. The authors thank Mr. Th. van Beneden for expert technical assistance.
REFERENCES Cran DG, Moor RM, Hay MF (1980): Fine structure of the sheep oocyte during antral follicle development. J Reprod Fert 59:125132. Critser ES, Leibfried-Rutledge ML, Eyestone WH, First NL (1986): Acquisition of developmental competence during maturation in vitro. Theriogenology 25:150. De Loos F, Van Vliet C, Van Maurik P, Kruip TAM (1989): Morphology of immature bovine oocytes. Gamete Res 24:197-204. Fayrer-Hosken RA, Younis AI, Brackett BG, McBridge CE, Harper KM, Keefer CL, Cabaniss DC (1988):Pregnancy after laparoscopic oviductal transfer of in vitro matured and in vitro fertilized bovine oocyks. Biol Reprod 38 (suppl 1):72/70. Fukui Y, Sakuma Y (1980): Maturation of bovine oocytes cultured in vitro: Relation to ovarian activity, follicular size and the presence or absence of cumulus cells. Biol Reprod 22669-673. Golbus MS, Stein MP (1976): Qualitative patterns of protein synthesis in the mouse oocyte. J Exp Zoo1 198337-342. Goto K, Kajihara Y,Kosaka S, Koba M, Nakanishi Y, Ogawa K (1988): Pregnancies after co-culture of cumulus cells with bovine embryos derived from in-vitro fertilization of in vitro matured follicular oocytes. J Reprod Fertil83:753-758. Kruip TAM, Cran DG, Van Beneden TH, Dieleman SJ (1983): Structural changes in bovine oocytes during final maturation in vivo. Gamete Res 829-47. Laemmli UK (1970): Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 227:680-685.
Leibfried L, First NL (1979): Characterization of bovine follicular oocytes and their ability to mature in vitro. J Anim Sci 48:76-86. Lu KH, Boland MP, Crosby TF, Gordon I (1987): In vitro fertilization of cattle oocytes matured in vitro. Theriogenology 27:251. Moor RM, Osborn JC, Cran DG, Walters DE (1981):Selective effect of gonadotrophins on cell coupling, nuclear maturation and protein synthesis in mammalian oocytes. J Embryo1 Exp Morphol61:347365. Sato E, Iritani A, Nishikawa Y (1977):Factors involved in maturation of pig and cattle follicular oocytes cultured in vitro. Jpn J Anim Reprod 23:12-18. Schultz RM, Wassarman PM (1977):Specific changes in the pattern of protein synthesis during meiotic maturation of mammalian oocytes in vitro. Proc Natl Acad Sci USA 74538-541. Staigmiller RN, Moor RM (1984): Effect of follicle cells on the maturation and developmental competence of ovine oocytes matured outside the follicle. Gamete Res 9:221-229. Stubbings RB, Betteridge KJ, Busrur PK (1988): Investigations of culture requirements for bovine oocyte maturation in vitro. Theriogenology 29:313. Sun FZ, Moor RM (1988): Nuclear cytoplasm interactions during ovine oocyte maturation. J Reprod Fertil Abstract Series 1:9/8. Utsumi K, Katoh H, Iritani A (1988):Developmental ability of bovine follicular oocytes matured in culture and fertilized in vitro. Theriogenology 29:320. Van Blerkom J, McGaughey RW (1978): Molecular differentiation of the rabbit ovum. I. During oocyte maturation in vivo and in vitro. Dev Biol 63:139-150. Xu KP, Greve T, Callesen H, Hyttel P (1987): Pregnancy resulting from cattle oocytes matured and fertilized in vitro. J Reprod Fertil 81:501-504. Younis AI, Brackett BG, Fayrer-Hosken RA (1989): Influence of serum and hormones on bovine oocyte maturation and fertilization in vitro. Gamete Res 23:189-201.
Analysis of Protein Synthesis in Morphologically Classified Bovine Follicular Oocytes Before and After Maturation In Vitro P.M.M. KASTROP,' M.M. BEVERS,' O.H.J. DESTRkE?
AND
TH.A.M. KRUIP'
'Department of Herd Health and Reproduction, Faculty of Veterinary Medicine, University of Utrecht, and 'Hubrecht Laboratory, The Netherlands Institute for Developmental Biology, Utrecht, The Netherlands
ABSTRACT Bovine cumulus oocyte complexes (COCs) were isolated from antral ovarian follicles (4-8 mm). Immature COCs were classified into four categories, based on the homogeneity and clearness of the ooplasm and the transparency and compactness of the cumulus investment. In this study, the incorporation of TCA-precipitable 35S-methionineand the protein synthesis patterns of oocytes of these four categories were examined. Before maturation in vitro, similar incorporation rates and identical protein synthesis patterns were observed between oocytes of categories 1-3. Immature oocytes of category 4 showed reduced incorporation rates and exhibited aberrant protein synthesis patterns. After maturation in vitro, the patterns of category 4 oocytes were identical with the patterns of those in categories 1-3. The incorporation of 35S-methionine into in vitro matured oocytes was lower ( P < .001) in all categories. Based on these results, it is concluded that the initial classification of oocytes into four categories can be reduced to two categories.
follicles between 4 and 8 mm, into four categories. The classification is based on morphological features of both the ooplasm and the cumulus investment. Previously, the ultrastructural characteristics and the capacity to mature in vitro of the four categories were investigated (De Loos et al., 1989). Although the morphological differences between categories 1-4 oocytes were confirmed at the ultrastructural level, they were not reflected in different maturation capacities. Only the degenerating category 4 oocytes exhibited a decreased capacity to mature in vitro (De Loos et al., 1989). In this study, protein synthesis patterns of individual oocytes of the four categories were analysed, both before and after in vitro maturation, to establish whether the observed morphological features are reflected in biochemical differences.
MATERIALS AND METHODS Collection and Culture of Cumulus Oocyte Complexes Bovine ovaries, obtained a t a local slaughterhouse, were rinsed and collected in Dulbecco's phosphate bufKey Words: Cow, Classification, Cumulus oocyte fered saline a t 30°C. Precautions were taken to avoid complexes, In vitro maturation bacterial contamination. The ovaries were transported to the laboratory within 1 h after slaughter. Antral follicles between 4 and 8 mm were punctured, and the INTRODUCTION follicular fluid aspirates were pooled in a conical cenThe percentage of oocytes which mature properly in trifuge tube. The settled COCs were recovered from the vitro and achieve developmental competence has been follicular fluid after 15 min of sedimentation and subclaimed to be related to both the size of the follicles, sequently washed in Dulbecco's phosphate buffered safrom which oocytes were recovered, and the presence of line, supplemented with 4 mg/ml bovine serum albua cumulus investment (Sato et al., 1977; Leibfried and min, 0.36 mM pyruvate, 23.8 mM lactate, and 5.5 mM First, 1979; Fukui and Sakuma, 1980; Staigmiller and glucose (medium A). COCs were classified under a steMoor, 1984; Critser et al., 1986; Younis et al., 1989). In reomicroscope (magnification 50 x ) and divided into spite of the application of these selection criteria, the four categories, based on the homogeneity and clearyield of advanced bovine preimplantation embryos is still quite low (Lu et al., 1987; Xu et al., 1987; Goto et al., 1988; Fayrer-Hosken et al., 1988; Stubbings et al., 1988; Utsumi et al., 1988). To improve this yield, reliable classification criteria will be necessary. September 26, 1989; accepted January 29, 1990. As an initial step towards a reliable and non-inva- Received Address reprint requests to P.M.M.Kastrop, Department of Herd sive classification system, we have distinguished im- Health and Reproduction, Faculty of Veterinary Medicine, University mature cumulus oocyte complexes (COCs), from antral of Utrecht, Yalelaan 7, 3584 CL Utrecht, The Netherlands. ~
~
0 1990 WILEY-LISS, INC.
~
PROTEIN SYNTHESIS IN BOVINE OOCYTES ness of the ooplasm and the transparency, integrity, and compactness of the cumulus investment (De Loos et al., 1989):Categories 1and 2 included COCs with a compact and multilayered cumulus investment and homogeneous ooplasm, although category 2 COCs exhibited a darker zone a t the periphery of the oocytes. Category 3 COCs possessed less compact and darker multilayered cumulus investments, whereas those of category 4 COCs were dark and irregularly expanded. The ooplasms in these COCs appeared irregular and contained dark clusters. The whole selection and classification procedure was carried out a t approximately 30°C and within 2-3 h after slaughter. Oocytes were matured in vitro by culture in Medium 199 with Earle's salts (Flow Laboratories, U.K.) supplemented with 10% heat-inactivated foetal calf serum, 1 pg/ml bLH, 2 pg/ml bFSH, and 1 kg/ml Oestradiol17p. Culture of COCs was carried out at 39°C in a humidified atmosphere of 5% C02 in air.
223
TABLE 1. Incorporation of "S-Methionine Into Bovine Oocyte Proteins, Before and After In Vitro Maturation (IVM) for the Four Morphological Categories "'S-methionine incoruoration (cpm/oocyte/36) Before N M After IVM
Category 1 2 3 4
9,118k 702 (15) 9,980 % 862 (12) 8,296 5 1,129 (12) 6,050 5 508 (12)
Values are mean rentheses.
f
5,661 2 296 (15) 6,138 f 522 (15) 3,748 t 284 (13) 3,292 f 428 (10)
S.E.M.for the number of oocytes in pa-
after in vitro maturation for 21 h in all four categories. Furthermore, category 4 oocytes exhibited a reduced incorporation (P 5 ,001) compared with categories 1-3 oocytes. The incorporation of 35S-methionine into the cumulus investment was about 20-50 times higher than was the incorporation into the oocytes (results not Radiolabelling and Electrophoresis shown), indicating the importance of a complete reCOCs were either radiolabelled immediately after moval of all cumulus cells. The patterns of de novo protein synthesis in individcollection for 3 h or cultured for 21 h, to accomplish in vitro maturation, and subsequently radiolabelled for 3 ual oocytes within the four categories are presented in h. Labelling was performed a t 39°C in medium A, con- Figure 1. Immature oocytes in categories 1-3 showed (specific activity > no differences in the protein synthesis patterns, taining 1mCi/ml ~-[~~Slmethionine 1,000 Ci/mmol; Amersham, U.K.). After labelling, the whereas category 4 oocytes exhibited an aberrant patoocytes were denuded of cumulus cells, and only intact tern (panel A). All the oocytes were in the germinal oocytes were collected and washed in medium A. These vesicle (GV)stage after the labelling period. The prooocytes were lysed individually by adding 20 kl SDS tein synthesis patterns of in vitro matured oocytes sample buffer (Laemmli, 19701, followed by heating at (panel B) showed no differences, i.e., the polypeptide 100°C for 2 min. Aliquots of 2 ~1 were used to deter- pattern of the in vitro matured category 4 oocyte was mine the amount of TCA-precipitable counts. The re- identical with those of the in vitro matured oocytes in mainder of each sample was stored at -20°C until elec- categories 1-3. The oocytes shown in panel B had extruded the first polar body after the 21 h culture and 3 trophoresis. Radiolabelled proteins were separated and resolved h labelling period. This indicated that nuclear matuby sodium dodecyl sulphate polyacrylamide gel electro- ration had been completed. Protein patterns of the immature oocytes in categophoresis (SDS-PAGE) according to the procedure of Laemmli (1970), using 8-15% linear gradient slab gels ries 1-3 (panel A), as compared to the in vitro matured of 0.75 mm thickness. Equal amounts of TCA-precipi- oocytes (panel B), showed marked differences, indicattable counts were loaded onto a gel. After electrophore- ing major changes in protein synthesis during meiotic sis, the gels were treated with AmplifyTM(Amersham, maturation. Protein synthesis patterns of both immaU.K.)for 30 min and dried under vacuum at 80°C. Sub- ture and in vitro matured oocytes in categories 1-3 sequently, the gels were exposed to Kodak-XAR5 films were always identical within a particular category. Also, no differences were observed between the patat -40°C for about 3 weeks. Mixtures of proteins with known molecular weight terns of in vitro matured oocytes in category 4 (results between 14.2 and 205 kDa (Sigma Chemicals Co., not shown). The heterogeneity within immature USA) were run simultaneously as standards and visu- oocytes in category 4 is shown in Figure 2. The majority of the protein synthesis patterns of these immature alized after electrophoresis by Coomassie staining. category 4 oocytes contained characteristic bands RESULTS which were also present in the polypeptide patterns of The incorporation of radiolabelled methionine into in vitro matured oocytes (lanes 2-5), but sometimes oocyte proteins was determined by TCA-precipitation, the polypeptide pattern resembled the pattern of imwhich enabled the loading of equal amounts of radio- mature oocytes in categories 1-3 (lane 1). activity per sample on a gel. The average incorporation DISCUSSION data of the four categories, both before and after in vitro maturation, are presented in Table 1.Analysis of In this study, we have investigated whether morphovariance revealed a decreased incorporation (P < .001) logical differences between immature COCs could be
224
P.M.M. KASTROP ET AL.
Fig. 1. Protein synthesis patterns of immature (A) and in vitro within a 21 h culture period (see Materials and Methods).The most matured (B)bovine oocytes. Categories 1-4 COCs were radiolabelled prominent differences between the patterns of immature categories with "S-methionine for 3 h and individually prepared for SDS-PAGE 1-3 and in vitro matured oocytes of categories 1-4 (116,95,70,54,33, and fluorography. In vitro maturation of oocytes was accomplished 26,22,and 14 kD)are marked by arrows in panel B.
confirmed at the level of protein synthetic capacities. As radiolabelling of newly synthesized proteins is dependent on amino acid incorporation, these data supplied additional information. The lower incorporation
rate in category 4 oocytes might be caused by a reduced number of junctional complexes. Ultrastructural investigations showed that category 4 oocytes possess less frequently occurring and not oocyte-penetrating junc-
PROTEIN SYNTHESIS IN BOVINE OOCYTES
Fig. 2. Protein synthesis patterns of five immature category 4 oocytes. Whereas the less frequently occurring pattern in lane 1 resembles the patterns of immature categories 1-3 oocytes (Fig. 1, panel A), the patterns in lanes 2-5 exhibited characteristics which were also observed in the patterns of in vitro matured oocytes (Fig. 1, panel B). COCs were radiolabelled for 3 h with '"S-methionine and individually prepared for SDS-PAGE and fluorography (see Materials and Methods).
tional complexes (De Loos et al., 1989). In addition, a regression of junctional complexes during maturation has been reported (Cran et al., 1980; Kruip et al.,
225
19831, which might explain the reduced incorporation after in vitro maturation. The protein synthesis patterns of both immature and in vitro matured oocytes in categories 1-4 did not reflect the observed morphological differences between the four categories. The majority of immature oocytes in category 4 exhibited a protein synthesis pattern which deviated from those in immature categories 1-3. This supports recently reported ultrastructural observations (De Loos et al., 19891, which indicated that category 4 consists of a heterogeneous population of degenerating oocytes. The differences between immature oocytes and in vitro matured oocytes in categories 1-3 revealed changes in protein synthesis during bovine oocyte maturation. Whether these changes occur subsequent to germinal vesicle breakdown (GVBD),as described for a number of other mammalian species (mouse: Golbus and Stein, 1976; Schultz and Wassarman, 1977; rabbit: Van Blerkom and McGaughey, 1978; sheep: Moor et al., 19811, is now under study. The remarkable resemblance of the protein synthesis patterns of most of the immature category 4 oocytes to the patterns of in vitro matured oocytes suggests an uncoupling of the nuclear and cytoplasmic maturation processes. This is supported by the data of Sun and Moor (19881, showing that the extensive changes in protein synthesis subsequent to GVBD is not dependent on transcription or the mixing of the karyoplasm with the cytoplasm after nuclear breakdown. A similar resemblance between these two groups was observed a t the ultrastructural level, as morphological characteristics of maturing oocytes were recognized in category 4 oocytes (De Loos et al., 1989). The uncoupling of nuclear and cytoplasmic maturation might be induced by the disturbed intercellular contact between cumulus cells and immature oocytes in category 4. Although morphological differences between oocyte categories, seen with light microscopy, were confirmed a t the ultrastructural level (De Loos et al., 19891, this study demonstrated that only category 4 differs obviously from categories 1-3 in protein synthesis patterns of the oocytes. Although a few immature oocytes in category 4 have protein synthesis patterns resembling those of immature oocytes in categories 1-3, the applied classification system distinguishes category 4 oocytes as heterogeneous and degenerating oocytes. Since these oocytes completed meiosis I a t a lower frequency in vitro (De Loos et al., 19891, they should be avoided in in vitro maturation and fertilization studies. So, the use of COCs with a compact and multilayered cumulus investment, i.e., categories 1-3 COCs, improves the proportion of oocytes which mature properly in vitro and achieve developmental competence. This confirms previous findings of Younis et al. (19891 and implies that based on these results categories 1-3 COCs may be pooled in practice for in vitro maturation and fertilization studies.
226
P.M.M. KASTROP ET AL.
ACKNOWLEDGMENTS This study was supported by grants of the Dutch Programme Committee for Agriculture Biotechnology. The authors thank Mr. Th. van Beneden for expert technical assistance.
REFERENCES Cran DG, Moor RM, Hay MF (1980): Fine structure of the sheep oocyte during antral follicle development. J Reprod Fert 59:125132. Critser ES, Leibfried-Rutledge ML, Eyestone WH, First NL (1986): Acquisition of developmental competence during maturation in vitro. Theriogenology 25:150. De Loos F, Van Vliet C, Van Maurik P, Kruip TAM (1989): Morphology of immature bovine oocytes. Gamete Res 24:197-204. Fayrer-Hosken RA, Younis AI, Brackett BG, McBridge CE, Harper KM, Keefer CL, Cabaniss DC (1988):Pregnancy after laparoscopic oviductal transfer of in vitro matured and in vitro fertilized bovine oocyks. Biol Reprod 38 (suppl 1):72/70. Fukui Y, Sakuma Y (1980): Maturation of bovine oocytes cultured in vitro: Relation to ovarian activity, follicular size and the presence or absence of cumulus cells. Biol Reprod 22669-673. Golbus MS, Stein MP (1976): Qualitative patterns of protein synthesis in the mouse oocyte. J Exp Zoo1 198337-342. Goto K, Kajihara Y,Kosaka S, Koba M, Nakanishi Y, Ogawa K (1988): Pregnancies after co-culture of cumulus cells with bovine embryos derived from in-vitro fertilization of in vitro matured follicular oocytes. J Reprod Fertil83:753-758. Kruip TAM, Cran DG, Van Beneden TH, Dieleman SJ (1983): Structural changes in bovine oocytes during final maturation in vivo. Gamete Res 829-47. Laemmli UK (1970): Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 227:680-685.
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