Breakdown of the Germinal Vesicle in Bovine Oocytes Cultivated In Vitro J. MOTLIK, H. H. KOEFOED-JOHNSEN I AND J. FULKA Czechoslovak Academy of Sciences, Institute of Animal Physiology and Genetics, Department of Genetics, 277 21 Libgchou, Czechoslovakia
ABSTRACT Breakdown of the germinal vesicle (GV) was studied in bovine follicular oocytes cultivated in vitro. The intact GV of control oocytes did not correspond to the findings in porcine oocytes (GV I). Instead it was characterized by a nuclear membrane and a few chromocenters in finely granular nucleoplasm (GV 11). After two and three hours of cultivation, GV I11 predominated (50% and 64% respectively). Faintly-staining chromatin and after a longer interval in the form of filaments also appeared round the chromocenters. After four hours, 35.8% of the oocytes were still a t stage GV I11 but the GV of 46.6% of the oocytes already contained filamentous bivalents (GV IV). After five hours, GV IV (59.7%) was typical, with a less distinct nuclear membrane and condensing bivalents. GV breakdown was completed in 30.4%of the oocytes after five hours of cultivation and in 92.5% after six hours. Since the initial observations of Edwards ('651, bovine follicular oocytes have been cultivated in vitro by Foote and Thibault ('691, Sreenan ('701, Hunter et al. ('72), Jagiello et al. ('74), Thibault et al. ('751, and Shea et al. ('76) with varying degrees of success. Oocytes cultivated in vitro can be penetrated after transfer to the bovine or porcine oviduct (Hunter et al., '72; Shea et al., '76). In agreement with observations in rabbits, pigs, and man, results show that bovine oocytes which have matured in vitro up to metaphase I1 do not transform the sperm head to a male pronucleus (Thibault e t al., '76). Usai and Yanagimachi ('76) convincingly demonstrated that the ability of the cytoplasm to decondense the sperm head appears together with the breakdown of the germinal vesicle (GV) i n hamster oocytes matured in vivo. Recently Wassarman and Letourneau ('76a,b) described RNA synthesis during GV dissolution and 3H-lysineincorporation into a variety of proteins that are partly concentrated in the GV and that become intimately associated with condensed chromosomes. From the available knowledge it seems obvious that this particular stage of development-the breakdown of the germinal vesicle-is very important for the acquisition of normal fertilizing ability and for further developmental capacit y of oocytes. J. EXP. ZOOL. (1978) 205: 377-384.
In the present study we attempted to follow the cytological changes during the initial phase of cattle oocyte maturation. These changes, together with biochemical analyses, may help to improve our understanding of this period of oocyte development. MATERIALS AND METHODS
In order to study the time sequence of GV breakdown, bovine oocytes were isolated by aspiring follicles (4 4-7 mm) from the ovaries of slaughtered heifers and cows. They were taken to the laboratory in their own follicular fluid maintained a t 37°C. Within one hour after aspiration of follicles the oocytes were pooled using a stereomicroscope, washed three times in medium, and control oocytes were fixed immediately. All the oocytes with compact cumulus were cultured at 38°C in 0.1 ml medium under paraffin oil in 5%CO, in air. The culture medium TC 199 (pH 7.4, mOsm 285) was the same as the one used by Motlik and Fulka ('74). Two intervals were studied in each experiment, and i n each case about 15 control oocytes were fixed. On completing cultivation, the oocytes were stripped of cumulus cells, mounted on slides, and fixed for 24 hours in acetic alcohol (1:3). ' The Royal Veterinary and Agricultural University, Institute for Animal Reproduction, 13 Bulowsvej, DK-1870 Copenhagen V, Denmark.
377
378
J MOTLIK, H H. KOEFOED-JOHNSEN AND J. FULKA
less than 8%of the control oocytes (fig. 2). Although only oocytes with a compact cumulus were used, 5%of them were a t the LD stage. The incidence of advanced stages of meiosis, which are associated with follicular atresia, was much higher in oocytes with an expanded cumulus, or, conversely, with no cumulus cells a t all. The first signs of GV breakdown were observed after only one hour's cultivation, a t which time clouds of readily staining chromatin appeared around the chromocenters in a quarter of the oocytes (fig. 3). One hour later, hakf the oocytes were a t stage GV 111. After three hours, 64%were a t this stage; a chromocenter remnant, with the first fine chromatin filaments, was characteristic of these oocytes (fig. 4), some of which (13%)had already progressed to stage GV IV. Four hours after commencing cultivation, oocytes a t stage GV IV predominated (46.6%); RESULTS in these, the chromocenters had disappeared, The rapid course of GV breakdown in but single filamentous bivalents could be oboocytes cultured in vitro can be seen in table 1. served (fig. 5). At this time 35.8% of the Of the total number of 553 oocytes, 104 served oocytes were still a t stage GV 111. After five hours the typical stage was GV as controls. The GV organization typical of porcine oocytes, designated as GV I, was not IV (59.7%), with a less distinct nuclear memobserved in any of the control bovine oocytes. brane and condensing bivalents (fig. 6, 7). In At the beginning of culture most of them were 30.4%of the oocytes the nuclear membrane already a t stage GV 11, in as much as they had already disappeared and the oocytes were were characterized by a distinct nuclear mem- classified as ED. Apart from marked condenbrane, finely granular nucleoplasm with a few sation of the bivalents, the ED stage of bovine chromocenters lying alongside the nuclear oocytes was characterized by the presence of membrane or concentrated in one area in the the rest of the nucleoplasm which had not yet merged with the cytoplasm (fig. 8). GV (fig. 1). Good visibility of the nucleolus with the After six hours, 79.2% of the oocytes had chosen techniques proved to be a problem in reached stage LD. The bivalents were at their bovine oocytes. A nucleolus-like structure was maximum shortness and were usually cirobserved, together with the chromocenters, in cularly arranged. GV breakdown was com-
One percent orcein (Loba-Chemie, Vienna) was prepared in 25%acetic acid only, and the preparations were differentiated in the same concentration. This acetic acid concentration, lower than the one originally used by Hunter and Polge, ('66) ensures better staining of the chromatin filaments in bovine oocytes and also results in better visibility of the nuclear membrane after staining. The oocytes were examined with both light and phase contrast microscopy. For the classification of GV, criteria identical to those used in the previous study on pig oocytes were employed. The techniques used enabled observation of changes in the nuclear membrane, as well as in chromatin and nucleolus, and allowed us to examine them during maturation to the four specified classes as previously described in detail by Motlik and Fulka ('76).
TABLE 1
Breakdown ofthe germinal uesicle in bouine oocytes cultiuated in uitro Stage of meiosis
'
Hours of culture
No of exp.
No. of oocytes
0 1 2 3 4 5 6 Total
7
104 36 57 100 120 82 54 553
I
1
2 3 3 3 2
GVI
Germinal vesicle GVII
99(90.4) 27(75.0) 28(49.1) 23(23.0) 10( 8.3) 7( 8.5) 1( 1.8)
GV 111
Diakinesis GV IV
ED
LD
5( 4.8) g(25.0) Zg(50.9) 64(64.0) 43(35.8) 1( 0.8)
13(13.0) 56(46.7) 49(59.7) 3( 5.5)
The figures in parentheses give the percentages of the total numbers of oocytes.
6( 5.0) 25(30.5) 8(14.8)
5( 4.2) 42(77.7)
BREAKDOWN OF BOVINE GERMINAL VESICLE IN VITRO
pleted in 30.4%of the oocytes after five hours cultivation and in 92.5% after six. DISCUSSION
The time course of nuclear maturation can be compared a t the first 6 hour interval with the data of Jagiello et al. ('74), who observed that the majority of bovine oocytes were in the germinal vesicle stage and that some were in late diplotene and diakinesis. The proportion of oocytes in diakinesis and metaphase I rises after eight hours cultivation. In our work after the six hour interval 79%of the oocytes were a t the LD stage. Sreenan ('70) recorded a preponderance of metaphase 1 and diakinesis after ten hours cultivation. In both the above studies, the oocytes were in metaphase I1 after 19 to 21 hours-which is in full agreement with the results of our own experiment (Motlik and Fulka, unpublished results). Breakdown of the bovine GV is usually complete after six hours cultivation. It thus differs significantly from the GV breakdown rate in pigs, in which 16 to 20 hours are needed to attain the same maturation level (Motlik and Fulka, '76). A t this time, when the nuclear membrane is broken down, the chromatin of porcine oocytes, as a rule, is organized in individual filamentous bivalents. At the same stage in cattle, we see highly condensed bivalents in the form of orcein-positive points. The presence of a nucleoplasm residue in the ED stage is interesting. In the vicinity of the bivalents a zone with the fine granulation can always be identified. There is no doubt that this is GV material which is still separated from surrounding cytoplasm. In contradistinction, Hunter et al. ('72), using the same method for evaluating oocytes as was used in the present study, described intact GV in more than 90%of the oocytes and found various arrangements of nucleoli and threads or lumps of chromatin. A visible nucleolus was not typical in the GV in our experiments, and even in positive cases its identification was very difficult. Taking into account all the oocytes classified as GV I1 and GV 111, nucleoli were observed in only 7.6% and 4.7%of oocytes, respectively. Baker and Franchi ('67) demonstrated a nucleolus in the diplotene stage of the bovine oocyte nucleolus using an electron microscope.
379
The effort to gain more detail connected with GV breakdown may lead to a better understanding of nuclear maturation. The period of oocyte development under study is also important for macromolecular syntheses which in later phases are responsible for the cytoplasm maturation, and for insuring growth of the male pronucleus after sperm penetration. The coupling of morphological and biochemical aspects of development may help to improve possibilities for oocyte maturation in vitro. LITERATURE CITED Baker, T. G., and L. L. Franchi 1967 The fine structure of chromosomes in bovine primordial oocytes. J. Reprod. Fert., 14: 511-513. Edwards, R. G. 1965 Maturation in vitro of mouse, sheep, cow, pig, rhesus monkey and human ovarian oocytes. Nature (London), 208: 349-351. Foote, W. D., andC. Thibault 1969 Recherche8 expbrimentales sur la maturation in vitro des ovocytes de truie e t de veau. Ann. Biol. Anim. Biochem. Biophys., 9: 329-349. Hunter, R. H.F., R. A. S. Lawson and L. E. A. Rowson 1972 Maturation, transplantation and fertilization of ovarian oocytes in cattle. J. Repr. Fert., 30: 325-328. Hunter, R. H. F., andC. Polge 1966 Maturationof follicular oocytes in the pig after injection of human chorionic gonadotrophin. J. Reprod. Fert., 12: 525-531. Jagiello, G. M., W. A. Miller, M. B. Ducayen and J. S. Lin 1974 Chiasma frequency and disjunctional behavior of ewe and cow oocytes matured in vitro. Biol. Repr., 10:
354-363. Motlik, J., and J. Fulka 1974 Fertilization of pig follicular oocytes cultivated in vitro. J. Reprod. Fert., 36: 235-237. 1976 Breakdown of the germinal vesicle in pig oocytes in vivo and in vitro. J. Exp. Zool., 198: 155-162. Shea, B. F., J. P. A. Latour, K. N. Bedirian and R. D. Baker 1976 Maturation in vitro and subsequent permeability of bovine follicular oocytes. J. Anim. Sci., 43: 809-815. Sreenan, J. 1970 In vitro maturation and attempted fertilization of cattle follicular oocytes. J. Agric. Sci., Camb., 75: 393-396. Thibault, C., M. Gerard and Y. Menezo 1975 Acquisition par l'ovocyte de lapine et de veau du facteur de dtkondensation du noyau du spermatozoide ftkondant (MPGF). Ann. Biol. Anim. Biochim. Biophys., 15: 705-714. 1976 Nuclear and cytoplasmic aspects of mammalian oocyte maturation in vitro in relation to follicle size and fertilization. Sperm Action. Prog. Reprod. Biol., 1: 233-240(Karger, Basel). Usui, N., and R. Yanagimachi 1976 Behavior of hamster sperm nuclei incorporated into eggs a t various stages of maturation, fertilization, and early development. J. U1trastruct. Res., 57: 276-288. Wassarman, P. M., and G. E. Letourneau 1976a RNA synthesis in fully-grown mouse oocytes. Nature (London), 261: 73-74.
1976b Meiotic maturation of mouse oocytes in vitro: Association of newly synthesized proteins with condensing chromosomes. J. Cell Sci., 20: 549-568.
X
1,200.
4
An wcyte with GV I11 fixed a t three hours of culture. The rest of chromocenters and the fine chromatin strains are characteristic of this stage. X 2,000.
3 Germinal vesicle (GV 111) of an oocyte isolated after two hours of culture. Nuclear membrane and finely granular nucleoplasma are still visible. Around the chromocenters clouds of readily-staining chromatin appeared. X 1,500.
2 An oocyte with GV I1 fixed a t one hour of culture. Arrow points to nucleolus.
1 The germinal vesicle (GV 11) of a control oocyte with distinctly visible nuclear membrane and few chromocenters in finely granular nucleoplasm. X 1,500.
EXPLANATION OF FIGURES
PLATE I
s
38 1
8a,b Two oocytes with the nuclei in early diakinesis recovered after five hours of culture. GV material (arrow) which is still separated from surrounding cytoplasm. X 1,500.
7 The condensed bivalents along the nuclear membrane which, after staining, is indistinct (GV 1V). An oocyte after five hours of culture. X 1,500.
6 The less distinct nuclear membrane (arrow) and shortening bivalents are typical for oocytes after five hours of culture (GV IV). X 1,500.
5 Germinal vesicle (GV IV) of an oocyte recovered after four hours of culture. Single filamentous bivalents fill up the whole GV. Nuclear membrane is out of focus. X 1,500.
EXPLANATION OF FIGURES
PLATE 2
N
0
a
E >
383
ABSTRACT Breakdown of the germinal vesicle (GV) was studied in bovine follicular oocytes cultivated in vitro. The intact GV of control oocytes did not correspond to the findings in porcine oocytes (GV I). Instead it was characterized by a nuclear membrane and a few chromocenters in finely granular nucleoplasm (GV 11). After two and three hours of cultivation, GV I11 predominated (50% and 64% respectively). Faintly-staining chromatin and after a longer interval in the form of filaments also appeared round the chromocenters. After four hours, 35.8% of the oocytes were still a t stage GV I11 but the GV of 46.6% of the oocytes already contained filamentous bivalents (GV IV). After five hours, GV IV (59.7%) was typical, with a less distinct nuclear membrane and condensing bivalents. GV breakdown was completed in 30.4%of the oocytes after five hours of cultivation and in 92.5% after six hours. Since the initial observations of Edwards ('651, bovine follicular oocytes have been cultivated in vitro by Foote and Thibault ('691, Sreenan ('701, Hunter et al. ('72), Jagiello et al. ('74), Thibault et al. ('751, and Shea et al. ('76) with varying degrees of success. Oocytes cultivated in vitro can be penetrated after transfer to the bovine or porcine oviduct (Hunter et al., '72; Shea et al., '76). In agreement with observations in rabbits, pigs, and man, results show that bovine oocytes which have matured in vitro up to metaphase I1 do not transform the sperm head to a male pronucleus (Thibault e t al., '76). Usai and Yanagimachi ('76) convincingly demonstrated that the ability of the cytoplasm to decondense the sperm head appears together with the breakdown of the germinal vesicle (GV) i n hamster oocytes matured in vivo. Recently Wassarman and Letourneau ('76a,b) described RNA synthesis during GV dissolution and 3H-lysineincorporation into a variety of proteins that are partly concentrated in the GV and that become intimately associated with condensed chromosomes. From the available knowledge it seems obvious that this particular stage of development-the breakdown of the germinal vesicle-is very important for the acquisition of normal fertilizing ability and for further developmental capacit y of oocytes. J. EXP. ZOOL. (1978) 205: 377-384.
In the present study we attempted to follow the cytological changes during the initial phase of cattle oocyte maturation. These changes, together with biochemical analyses, may help to improve our understanding of this period of oocyte development. MATERIALS AND METHODS
In order to study the time sequence of GV breakdown, bovine oocytes were isolated by aspiring follicles (4 4-7 mm) from the ovaries of slaughtered heifers and cows. They were taken to the laboratory in their own follicular fluid maintained a t 37°C. Within one hour after aspiration of follicles the oocytes were pooled using a stereomicroscope, washed three times in medium, and control oocytes were fixed immediately. All the oocytes with compact cumulus were cultured at 38°C in 0.1 ml medium under paraffin oil in 5%CO, in air. The culture medium TC 199 (pH 7.4, mOsm 285) was the same as the one used by Motlik and Fulka ('74). Two intervals were studied in each experiment, and i n each case about 15 control oocytes were fixed. On completing cultivation, the oocytes were stripped of cumulus cells, mounted on slides, and fixed for 24 hours in acetic alcohol (1:3). ' The Royal Veterinary and Agricultural University, Institute for Animal Reproduction, 13 Bulowsvej, DK-1870 Copenhagen V, Denmark.
377
378
J MOTLIK, H H. KOEFOED-JOHNSEN AND J. FULKA
less than 8%of the control oocytes (fig. 2). Although only oocytes with a compact cumulus were used, 5%of them were a t the LD stage. The incidence of advanced stages of meiosis, which are associated with follicular atresia, was much higher in oocytes with an expanded cumulus, or, conversely, with no cumulus cells a t all. The first signs of GV breakdown were observed after only one hour's cultivation, a t which time clouds of readily staining chromatin appeared around the chromocenters in a quarter of the oocytes (fig. 3). One hour later, hakf the oocytes were a t stage GV 111. After three hours, 64%were a t this stage; a chromocenter remnant, with the first fine chromatin filaments, was characteristic of these oocytes (fig. 4), some of which (13%)had already progressed to stage GV IV. Four hours after commencing cultivation, oocytes a t stage GV IV predominated (46.6%); RESULTS in these, the chromocenters had disappeared, The rapid course of GV breakdown in but single filamentous bivalents could be oboocytes cultured in vitro can be seen in table 1. served (fig. 5). At this time 35.8% of the Of the total number of 553 oocytes, 104 served oocytes were still a t stage GV 111. After five hours the typical stage was GV as controls. The GV organization typical of porcine oocytes, designated as GV I, was not IV (59.7%), with a less distinct nuclear memobserved in any of the control bovine oocytes. brane and condensing bivalents (fig. 6, 7). In At the beginning of culture most of them were 30.4%of the oocytes the nuclear membrane already a t stage GV 11, in as much as they had already disappeared and the oocytes were were characterized by a distinct nuclear mem- classified as ED. Apart from marked condenbrane, finely granular nucleoplasm with a few sation of the bivalents, the ED stage of bovine chromocenters lying alongside the nuclear oocytes was characterized by the presence of membrane or concentrated in one area in the the rest of the nucleoplasm which had not yet merged with the cytoplasm (fig. 8). GV (fig. 1). Good visibility of the nucleolus with the After six hours, 79.2% of the oocytes had chosen techniques proved to be a problem in reached stage LD. The bivalents were at their bovine oocytes. A nucleolus-like structure was maximum shortness and were usually cirobserved, together with the chromocenters, in cularly arranged. GV breakdown was com-
One percent orcein (Loba-Chemie, Vienna) was prepared in 25%acetic acid only, and the preparations were differentiated in the same concentration. This acetic acid concentration, lower than the one originally used by Hunter and Polge, ('66) ensures better staining of the chromatin filaments in bovine oocytes and also results in better visibility of the nuclear membrane after staining. The oocytes were examined with both light and phase contrast microscopy. For the classification of GV, criteria identical to those used in the previous study on pig oocytes were employed. The techniques used enabled observation of changes in the nuclear membrane, as well as in chromatin and nucleolus, and allowed us to examine them during maturation to the four specified classes as previously described in detail by Motlik and Fulka ('76).
TABLE 1
Breakdown ofthe germinal uesicle in bouine oocytes cultiuated in uitro Stage of meiosis
'
Hours of culture
No of exp.
No. of oocytes
0 1 2 3 4 5 6 Total
7
104 36 57 100 120 82 54 553
I
1
2 3 3 3 2
GVI
Germinal vesicle GVII
99(90.4) 27(75.0) 28(49.1) 23(23.0) 10( 8.3) 7( 8.5) 1( 1.8)
GV 111
Diakinesis GV IV
ED
LD
5( 4.8) g(25.0) Zg(50.9) 64(64.0) 43(35.8) 1( 0.8)
13(13.0) 56(46.7) 49(59.7) 3( 5.5)
The figures in parentheses give the percentages of the total numbers of oocytes.
6( 5.0) 25(30.5) 8(14.8)
5( 4.2) 42(77.7)
BREAKDOWN OF BOVINE GERMINAL VESICLE IN VITRO
pleted in 30.4%of the oocytes after five hours cultivation and in 92.5% after six. DISCUSSION
The time course of nuclear maturation can be compared a t the first 6 hour interval with the data of Jagiello et al. ('74), who observed that the majority of bovine oocytes were in the germinal vesicle stage and that some were in late diplotene and diakinesis. The proportion of oocytes in diakinesis and metaphase I rises after eight hours cultivation. In our work after the six hour interval 79%of the oocytes were a t the LD stage. Sreenan ('70) recorded a preponderance of metaphase 1 and diakinesis after ten hours cultivation. In both the above studies, the oocytes were in metaphase I1 after 19 to 21 hours-which is in full agreement with the results of our own experiment (Motlik and Fulka, unpublished results). Breakdown of the bovine GV is usually complete after six hours cultivation. It thus differs significantly from the GV breakdown rate in pigs, in which 16 to 20 hours are needed to attain the same maturation level (Motlik and Fulka, '76). A t this time, when the nuclear membrane is broken down, the chromatin of porcine oocytes, as a rule, is organized in individual filamentous bivalents. At the same stage in cattle, we see highly condensed bivalents in the form of orcein-positive points. The presence of a nucleoplasm residue in the ED stage is interesting. In the vicinity of the bivalents a zone with the fine granulation can always be identified. There is no doubt that this is GV material which is still separated from surrounding cytoplasm. In contradistinction, Hunter et al. ('72), using the same method for evaluating oocytes as was used in the present study, described intact GV in more than 90%of the oocytes and found various arrangements of nucleoli and threads or lumps of chromatin. A visible nucleolus was not typical in the GV in our experiments, and even in positive cases its identification was very difficult. Taking into account all the oocytes classified as GV I1 and GV 111, nucleoli were observed in only 7.6% and 4.7%of oocytes, respectively. Baker and Franchi ('67) demonstrated a nucleolus in the diplotene stage of the bovine oocyte nucleolus using an electron microscope.
379
The effort to gain more detail connected with GV breakdown may lead to a better understanding of nuclear maturation. The period of oocyte development under study is also important for macromolecular syntheses which in later phases are responsible for the cytoplasm maturation, and for insuring growth of the male pronucleus after sperm penetration. The coupling of morphological and biochemical aspects of development may help to improve possibilities for oocyte maturation in vitro. LITERATURE CITED Baker, T. G., and L. L. Franchi 1967 The fine structure of chromosomes in bovine primordial oocytes. J. Reprod. Fert., 14: 511-513. Edwards, R. G. 1965 Maturation in vitro of mouse, sheep, cow, pig, rhesus monkey and human ovarian oocytes. Nature (London), 208: 349-351. Foote, W. D., andC. Thibault 1969 Recherche8 expbrimentales sur la maturation in vitro des ovocytes de truie e t de veau. Ann. Biol. Anim. Biochem. Biophys., 9: 329-349. Hunter, R. H.F., R. A. S. Lawson and L. E. A. Rowson 1972 Maturation, transplantation and fertilization of ovarian oocytes in cattle. J. Repr. Fert., 30: 325-328. Hunter, R. H. F., andC. Polge 1966 Maturationof follicular oocytes in the pig after injection of human chorionic gonadotrophin. J. Reprod. Fert., 12: 525-531. Jagiello, G. M., W. A. Miller, M. B. Ducayen and J. S. Lin 1974 Chiasma frequency and disjunctional behavior of ewe and cow oocytes matured in vitro. Biol. Repr., 10:
354-363. Motlik, J., and J. Fulka 1974 Fertilization of pig follicular oocytes cultivated in vitro. J. Reprod. Fert., 36: 235-237. 1976 Breakdown of the germinal vesicle in pig oocytes in vivo and in vitro. J. Exp. Zool., 198: 155-162. Shea, B. F., J. P. A. Latour, K. N. Bedirian and R. D. Baker 1976 Maturation in vitro and subsequent permeability of bovine follicular oocytes. J. Anim. Sci., 43: 809-815. Sreenan, J. 1970 In vitro maturation and attempted fertilization of cattle follicular oocytes. J. Agric. Sci., Camb., 75: 393-396. Thibault, C., M. Gerard and Y. Menezo 1975 Acquisition par l'ovocyte de lapine et de veau du facteur de dtkondensation du noyau du spermatozoide ftkondant (MPGF). Ann. Biol. Anim. Biochim. Biophys., 15: 705-714. 1976 Nuclear and cytoplasmic aspects of mammalian oocyte maturation in vitro in relation to follicle size and fertilization. Sperm Action. Prog. Reprod. Biol., 1: 233-240(Karger, Basel). Usui, N., and R. Yanagimachi 1976 Behavior of hamster sperm nuclei incorporated into eggs a t various stages of maturation, fertilization, and early development. J. U1trastruct. Res., 57: 276-288. Wassarman, P. M., and G. E. Letourneau 1976a RNA synthesis in fully-grown mouse oocytes. Nature (London), 261: 73-74.
1976b Meiotic maturation of mouse oocytes in vitro: Association of newly synthesized proteins with condensing chromosomes. J. Cell Sci., 20: 549-568.
X
1,200.
4
An wcyte with GV I11 fixed a t three hours of culture. The rest of chromocenters and the fine chromatin strains are characteristic of this stage. X 2,000.
3 Germinal vesicle (GV 111) of an oocyte isolated after two hours of culture. Nuclear membrane and finely granular nucleoplasma are still visible. Around the chromocenters clouds of readily-staining chromatin appeared. X 1,500.
2 An oocyte with GV I1 fixed a t one hour of culture. Arrow points to nucleolus.
1 The germinal vesicle (GV 11) of a control oocyte with distinctly visible nuclear membrane and few chromocenters in finely granular nucleoplasm. X 1,500.
EXPLANATION OF FIGURES
PLATE I
s
38 1
8a,b Two oocytes with the nuclei in early diakinesis recovered after five hours of culture. GV material (arrow) which is still separated from surrounding cytoplasm. X 1,500.
7 The condensed bivalents along the nuclear membrane which, after staining, is indistinct (GV 1V). An oocyte after five hours of culture. X 1,500.
6 The less distinct nuclear membrane (arrow) and shortening bivalents are typical for oocytes after five hours of culture (GV IV). X 1,500.
5 Germinal vesicle (GV IV) of an oocyte recovered after four hours of culture. Single filamentous bivalents fill up the whole GV. Nuclear membrane is out of focus. X 1,500.
EXPLANATION OF FIGURES
PLATE 2
N
0
a
E >
383
