- Email: [email protected]
Procedures for induction of spawning and meiotic maturation of starfish oocytes by treatment with 1-methyladenine
482
Margaret
Stevens
PROCEDURES FOR INDUCTION OF SPAWNING AND MEIOTIC MATURATION OF STARFISH OOCYTES BY TREATMENT WITH I-METHYLADENINE MARGARET Stanford
STEVENS, Hopkins
University,
Pacific
Grove,
Marine Station of Calif. 93950, USA
A polypeptide released from the radial nerve (radial nerve factor or gonad stimulating substance) induces shedding of gametes in starfish [l, 21. In the female, this factor stimulates the ovary wall to produce a meiosisinducing substance. This substance, which has been identified as I-methyladenine [8], mediates both spawning and maturation of oocytes [5, 6, 7, 91. I here describe methods for the use of I-methyladenine (I-MeAde) to induce spawning, meiotic maturation, and subsequent development in five species of California starfish. My results confirm observations by Kanatani on the nature of the mechanism of spawning. To examine spawning responses, ovarian fragments 2-3 mm long were washed in sea water to remove loose oocytes and then placed in small volumes (0.1-1.0 ml) of 1.3 X 1O-4 M I-MeAde (Sigma Chemicals) dissolved in sea water. To observe oocyte maturation, ovaries were torn with forceps, and the oocytes were released by agitating the tissue with a pipette. In all the species included in this study, oocytes isolated in such a manner do not mature in sea water unless I-MeAde is present. Six species of asteroids, including Patiria miniata, Pisaster brevispinus, Pisaster giganteus, Pisaster ochraceus, Pycnopodia helianthoides, and Mediaster aequalis, were exam-
ined for spawning of ovarian fragments, in addition to the echinoid Dendraster excentricus and the holothuroid Stichopus californicus. The non-asteroids, although ripe, did not spawn. Spawning, however, occurred in all the starfish. More extensive experiments Exptl
Cell Res 59
with P. miniata showed that concentrations as low as 1 x 1O-6 M are fully effective in inducing spawning. Spawning begins in ovarian fragments after 20-30 min incubation in l-MeAde and continues for 5-10 min. Using several species of starfish, Kanatani [5, 6, 71, has shown that spawning involves a reduction of intra-ovarian adhesivity due to the loss of the follicles surrounding the oocytes. These follicles are composed of numerous cells forming a hollow sphere around each oocyte. In the intact ovary each follicle adheres closely to the follicles of adjacent oocytes. I-MeAde appears to result in dissolution of the cementing substance between the follicle cells and thus in loosening of the oocytes. Kanatani postulates that this loosening of contacts, combined with the tension already present in the ovary wall, is sufficient to force the oocytes out of the ovary. The oocytes of the starfish used in the present study are also surrounded by follicles which dissolved as a result of treatment with I-MeAde. In isolated oocytes of P. miniata dissolution occurs 20-30 min after addition of 1-MeAde, which is similar to the time required for I-MeAde-induced spawning to occur. Isolated oocytes incubated in calcium-free sea water for 1 h also lose their follicles. However, ovarian fragments do not spawn in calcium-free sea water, presumably because calcium is necessary to maintain the contracted state of the ovary wall [5]. If such fragments are first treated with calcium-free sea water long enough (45 min) to disintegrate the follicles and are then returned to normal sea water, spawning occurs in the absence of 1-MeAde. These findings confirm the results of similar experiments performed by Kanatani and support the mechanism of spawning that he has proposed [5]. They also indicate that this phenomenon may be widespread among starfish.
Spawning and meiosis in starfish
Table 1. Germinal vesicle breakdown at various concentrations of I-methyladenine
483
Table
3. Relationship qf germinal vesicle breakdown (g.v.) to fertilizability in Patiria miniata oocytes
% germinal vesicle breakdown Cont. 1-MeAde W/l) Sea water control 1.3 x lo-* 1.3 x lo-’ 1.3 x 10-S 1.3 x10-6 1.3 x 10-4
Patiria miniata
17 28 92 100 100 100
Pisaster breoispinus
10 13 i: 97 95
Pisaster ochra-
ceus
17 24 90 88 88 91
Pycnopodia helianthoides
5 7 99 100 100 100
Oocytes were incubated 45 min in 1-MeAde in sea water. Percentages of germinal vesicle breakdown were determined microscopically.
1-MeAde also induced meiosis in the oocytes of five species of starfish examined (P. miniata, P. brevispinus, P. giganteus, P. ochraceus, and P. helianthoides) at concentrations
as low as 1.3 x lo-’ M (table 1). At 15°C meiotic events occur in reasonable synchrony, as is reported in table 2. Twenty-five to 28 min after addition of 1-MeAde, germinal vesicle breakdown first becomes apparent as a waviness of the nuclear outline. Several minutes later the nucleolus disappears, and the nucleus begins to shrink. At 34-35 min after addition of 1-MeAde the nuclear membrane dissolves, and by 45 min no nucleus is apparent. These events were examined in detail in oocytes of Table 2. Timing of meiotic events in oocytes of Patiria miniata at 15°C Time in I-MeAde (min)
State of maturation
0 25-28 30-32 34-35 4047 95-l 10 150-165
1-MeAde added Nuclear outline wavy Nucleolus disappears; nucleus shrinks Nuclear membrane gone No nuclear spot apparent First polar body Second polar body
Minutes of incubation
% g.v. breakdown 30 set aliquot
% Fertilization
0 15 20 25
10 16 17 40
9 10 29 38
it 40
100 98 100
99 98 100
Oocytes were incubated in 1.3 x 1O-5 M 1-MeAde. The incubation times were arranged so that all samples were fertilized simultaneously. Thirty seconds before addition of sperm and again 5 min after sperm addition, aliquots were removed from each sample and fixed in 5 % formaldehyde in sea water. The 30set aliquot was examined microscopically to determine the percentage of maturing occytes. Shrinkage and irregularity of the nucleus were used as criteria for germinal vesicle breakdown. The 5-min aliquot was examined to determine for each sample the percentage of fertilized oocytes, as indicated by elevation of the fertilization membrane.
P. miniata and P. helianthoides. Preliminary observations indicate that the other species follow much the same pattern. Delage [3] has shown that germinal vesicle breakdown must occur before starfish oocytes can be fertilized. My observations also show a chronological correlation between germinal vesicle breakdown and fertilizability in lMeAde-treated oocytes. In the experiment summarized in table 3, samples were incubated for varying lengths of time before addition of sperm. An aliquot from each sample was fixed 30 set before and 5 min after sperm addition. The percentage of maturing oocytes was determined in the 30-second aliquot, and the percentage of fertilized oocytes was counted in the second aliquot. As can be seen from table 3, the greatest percentage of oocytes became fertilizable between 25 and 30 min, when the nucleus is exhibiting the first signs of breakdown. Microscopic obserExptl Cell Res 59
484
J. M. R. Hatfield et al.
vations also indicate that although germinal vesicle breakdown is required, the complete disappearance of the nuclear membrane is not necessary for oocytes to be fertilizable. The results of these experiments show that 1-MeAde mediates both spawning and maturation of the oocyte. Spawning involves dissolution of the follicle surrounding each oocyte. Follicle dissolution occurs in both ovarian fragments and isolated oocytes after approximately the same incubation time. IMethyladenine-initiated meiosis occurs in ovaries, ovarian fragments, and isolated oocytes. From these results the following method has been developed for obtaining starfish oocytes which yield consistently high percentages of fertilization and of normal larvae. Ovary fragments or isolated oocytes are incubated in 1 x 1O-6 M I-MeAde in sea water. Testes are removed from ripe male starfish and stored in a cool place until needed. Just before fertilization several milliliters of sea water are added to the dry testes, and the sperms are released by agitation. One drop of the resulting sperm suspension is sufficient to fertilize several milliliters of oocytes. At 15°C the oocytes are then washed to remove excess sperm and allowed to develop in a cool place. Concentrations of I-MeAde as high as 1 x 1O-3 M can be used without ill effects. A certain number of immature oocytes are unavoidable, but the number can be greatly reduced by treating ovarian fragments and collecting only those oocytes that are shed. lMeAde does not need to be present at the time of fertilization, as it neither activates nor inhibits the sperm. I-MeAde can also be used for biochemical studies of meiosis, as it provides a simple way to synchronize the meiotic events of large numbers of oocytes. Studies concerning the biochemical events of meiosis are currently underway in this laboratory. Exptl CeN Res 59
I thank Dr David Epel for his advice concerning the work reported in this paper and for his criticism of the manuscript. I also thank Dr Haruo Kanatani for a stimulating discussion of this work. Supported by NIH Eredoctoral Fellowship (5 FOl GM41249-02) from the General Medical Sciences Institute and’by NSF grant GB-8002.
REFERENCES 1. 2. 3. 4. 2: I. 8. 9.
Chaet, A B, Biol bull 126 (1964) 8. -Ibid 130 (1966) 43. Delage, Y, Arch zoo1 exptl gen (ser 3) 8 (1901) 284. Kanatani, H, Science 146 (1964) 1177. - Gen camp endocrinology, suppl. 2 (1969) 582. - Exptl cell res 57 (1969) 333. Kanatani. H & Ohnuri. M. Biol bull 131 (1966) 104. Kanatani; H, ShGai, ‘H,‘Nakanishi, K & Kurokawa, T, Nature 221 (1969) 273. Schuetz, A W & Biggers, J D, Exptl cell res 46 (1967) 624.
Received October 21, 1969 Revised version received December 12, 1969
MEASUREMENT OF THE ULTRAVIOLET ABSORPTION IN SPECIFIC PARTS OF BOTH LIVING AND FIXED MAMMALIAN CELLS, USING A SPECIALLY DESIGNED MICROSPECTROPHOTOMETER J. M. R. HATFIELD, L. SCHULZE, and D. ERNST, Department of Radiotherapeutics, University of Cambridge, UK, and Institut fiir Strahlenbiologie, Technische Universitiit, Hannover, W. Germany
Summary Measurements of UV absorption by different parts of living Hep/2 cells have been obtained using a microspectrophotometer employing a low intensity of UV light. Between 255 nm and 280 nm, the nucleolus, nuclear sap and cytoplasm show significantly different absorptions. Living cells and cells fixed by freeze substitution show a similar pattern of UV absorption, although the absolute values may differ slightly.
The purpose of this work was to investigate the absorption of ultraviolet (UV) light by the nucleolus, nuclear sap, and cytoplasm of mammalian cells in tissue culture. This information is necessary to relate the effect of UV radiation on living cells, to the doses ab-
Margaret
Stevens
PROCEDURES FOR INDUCTION OF SPAWNING AND MEIOTIC MATURATION OF STARFISH OOCYTES BY TREATMENT WITH I-METHYLADENINE MARGARET Stanford
STEVENS, Hopkins
University,
Pacific
Grove,
Marine Station of Calif. 93950, USA
A polypeptide released from the radial nerve (radial nerve factor or gonad stimulating substance) induces shedding of gametes in starfish [l, 21. In the female, this factor stimulates the ovary wall to produce a meiosisinducing substance. This substance, which has been identified as I-methyladenine [8], mediates both spawning and maturation of oocytes [5, 6, 7, 91. I here describe methods for the use of I-methyladenine (I-MeAde) to induce spawning, meiotic maturation, and subsequent development in five species of California starfish. My results confirm observations by Kanatani on the nature of the mechanism of spawning. To examine spawning responses, ovarian fragments 2-3 mm long were washed in sea water to remove loose oocytes and then placed in small volumes (0.1-1.0 ml) of 1.3 X 1O-4 M I-MeAde (Sigma Chemicals) dissolved in sea water. To observe oocyte maturation, ovaries were torn with forceps, and the oocytes were released by agitating the tissue with a pipette. In all the species included in this study, oocytes isolated in such a manner do not mature in sea water unless I-MeAde is present. Six species of asteroids, including Patiria miniata, Pisaster brevispinus, Pisaster giganteus, Pisaster ochraceus, Pycnopodia helianthoides, and Mediaster aequalis, were exam-
ined for spawning of ovarian fragments, in addition to the echinoid Dendraster excentricus and the holothuroid Stichopus californicus. The non-asteroids, although ripe, did not spawn. Spawning, however, occurred in all the starfish. More extensive experiments Exptl
Cell Res 59
with P. miniata showed that concentrations as low as 1 x 1O-6 M are fully effective in inducing spawning. Spawning begins in ovarian fragments after 20-30 min incubation in l-MeAde and continues for 5-10 min. Using several species of starfish, Kanatani [5, 6, 71, has shown that spawning involves a reduction of intra-ovarian adhesivity due to the loss of the follicles surrounding the oocytes. These follicles are composed of numerous cells forming a hollow sphere around each oocyte. In the intact ovary each follicle adheres closely to the follicles of adjacent oocytes. I-MeAde appears to result in dissolution of the cementing substance between the follicle cells and thus in loosening of the oocytes. Kanatani postulates that this loosening of contacts, combined with the tension already present in the ovary wall, is sufficient to force the oocytes out of the ovary. The oocytes of the starfish used in the present study are also surrounded by follicles which dissolved as a result of treatment with I-MeAde. In isolated oocytes of P. miniata dissolution occurs 20-30 min after addition of 1-MeAde, which is similar to the time required for I-MeAde-induced spawning to occur. Isolated oocytes incubated in calcium-free sea water for 1 h also lose their follicles. However, ovarian fragments do not spawn in calcium-free sea water, presumably because calcium is necessary to maintain the contracted state of the ovary wall [5]. If such fragments are first treated with calcium-free sea water long enough (45 min) to disintegrate the follicles and are then returned to normal sea water, spawning occurs in the absence of 1-MeAde. These findings confirm the results of similar experiments performed by Kanatani and support the mechanism of spawning that he has proposed [5]. They also indicate that this phenomenon may be widespread among starfish.
Spawning and meiosis in starfish
Table 1. Germinal vesicle breakdown at various concentrations of I-methyladenine
483
Table
3. Relationship qf germinal vesicle breakdown (g.v.) to fertilizability in Patiria miniata oocytes
% germinal vesicle breakdown Cont. 1-MeAde W/l) Sea water control 1.3 x lo-* 1.3 x lo-’ 1.3 x 10-S 1.3 x10-6 1.3 x 10-4
Patiria miniata
17 28 92 100 100 100
Pisaster breoispinus
10 13 i: 97 95
Pisaster ochra-
ceus
17 24 90 88 88 91
Pycnopodia helianthoides
5 7 99 100 100 100
Oocytes were incubated 45 min in 1-MeAde in sea water. Percentages of germinal vesicle breakdown were determined microscopically.
1-MeAde also induced meiosis in the oocytes of five species of starfish examined (P. miniata, P. brevispinus, P. giganteus, P. ochraceus, and P. helianthoides) at concentrations
as low as 1.3 x lo-’ M (table 1). At 15°C meiotic events occur in reasonable synchrony, as is reported in table 2. Twenty-five to 28 min after addition of 1-MeAde, germinal vesicle breakdown first becomes apparent as a waviness of the nuclear outline. Several minutes later the nucleolus disappears, and the nucleus begins to shrink. At 34-35 min after addition of 1-MeAde the nuclear membrane dissolves, and by 45 min no nucleus is apparent. These events were examined in detail in oocytes of Table 2. Timing of meiotic events in oocytes of Patiria miniata at 15°C Time in I-MeAde (min)
State of maturation
0 25-28 30-32 34-35 4047 95-l 10 150-165
1-MeAde added Nuclear outline wavy Nucleolus disappears; nucleus shrinks Nuclear membrane gone No nuclear spot apparent First polar body Second polar body
Minutes of incubation
% g.v. breakdown 30 set aliquot
% Fertilization
0 15 20 25
10 16 17 40
9 10 29 38
it 40
100 98 100
99 98 100
Oocytes were incubated in 1.3 x 1O-5 M 1-MeAde. The incubation times were arranged so that all samples were fertilized simultaneously. Thirty seconds before addition of sperm and again 5 min after sperm addition, aliquots were removed from each sample and fixed in 5 % formaldehyde in sea water. The 30set aliquot was examined microscopically to determine the percentage of maturing occytes. Shrinkage and irregularity of the nucleus were used as criteria for germinal vesicle breakdown. The 5-min aliquot was examined to determine for each sample the percentage of fertilized oocytes, as indicated by elevation of the fertilization membrane.
P. miniata and P. helianthoides. Preliminary observations indicate that the other species follow much the same pattern. Delage [3] has shown that germinal vesicle breakdown must occur before starfish oocytes can be fertilized. My observations also show a chronological correlation between germinal vesicle breakdown and fertilizability in lMeAde-treated oocytes. In the experiment summarized in table 3, samples were incubated for varying lengths of time before addition of sperm. An aliquot from each sample was fixed 30 set before and 5 min after sperm addition. The percentage of maturing oocytes was determined in the 30-second aliquot, and the percentage of fertilized oocytes was counted in the second aliquot. As can be seen from table 3, the greatest percentage of oocytes became fertilizable between 25 and 30 min, when the nucleus is exhibiting the first signs of breakdown. Microscopic obserExptl Cell Res 59
484
J. M. R. Hatfield et al.
vations also indicate that although germinal vesicle breakdown is required, the complete disappearance of the nuclear membrane is not necessary for oocytes to be fertilizable. The results of these experiments show that 1-MeAde mediates both spawning and maturation of the oocyte. Spawning involves dissolution of the follicle surrounding each oocyte. Follicle dissolution occurs in both ovarian fragments and isolated oocytes after approximately the same incubation time. IMethyladenine-initiated meiosis occurs in ovaries, ovarian fragments, and isolated oocytes. From these results the following method has been developed for obtaining starfish oocytes which yield consistently high percentages of fertilization and of normal larvae. Ovary fragments or isolated oocytes are incubated in 1 x 1O-6 M I-MeAde in sea water. Testes are removed from ripe male starfish and stored in a cool place until needed. Just before fertilization several milliliters of sea water are added to the dry testes, and the sperms are released by agitation. One drop of the resulting sperm suspension is sufficient to fertilize several milliliters of oocytes. At 15°C the oocytes are then washed to remove excess sperm and allowed to develop in a cool place. Concentrations of I-MeAde as high as 1 x 1O-3 M can be used without ill effects. A certain number of immature oocytes are unavoidable, but the number can be greatly reduced by treating ovarian fragments and collecting only those oocytes that are shed. lMeAde does not need to be present at the time of fertilization, as it neither activates nor inhibits the sperm. I-MeAde can also be used for biochemical studies of meiosis, as it provides a simple way to synchronize the meiotic events of large numbers of oocytes. Studies concerning the biochemical events of meiosis are currently underway in this laboratory. Exptl CeN Res 59
I thank Dr David Epel for his advice concerning the work reported in this paper and for his criticism of the manuscript. I also thank Dr Haruo Kanatani for a stimulating discussion of this work. Supported by NIH Eredoctoral Fellowship (5 FOl GM41249-02) from the General Medical Sciences Institute and’by NSF grant GB-8002.
REFERENCES 1. 2. 3. 4. 2: I. 8. 9.
Chaet, A B, Biol bull 126 (1964) 8. -Ibid 130 (1966) 43. Delage, Y, Arch zoo1 exptl gen (ser 3) 8 (1901) 284. Kanatani, H, Science 146 (1964) 1177. - Gen camp endocrinology, suppl. 2 (1969) 582. - Exptl cell res 57 (1969) 333. Kanatani. H & Ohnuri. M. Biol bull 131 (1966) 104. Kanatani; H, ShGai, ‘H,‘Nakanishi, K & Kurokawa, T, Nature 221 (1969) 273. Schuetz, A W & Biggers, J D, Exptl cell res 46 (1967) 624.
Received October 21, 1969 Revised version received December 12, 1969
MEASUREMENT OF THE ULTRAVIOLET ABSORPTION IN SPECIFIC PARTS OF BOTH LIVING AND FIXED MAMMALIAN CELLS, USING A SPECIALLY DESIGNED MICROSPECTROPHOTOMETER J. M. R. HATFIELD, L. SCHULZE, and D. ERNST, Department of Radiotherapeutics, University of Cambridge, UK, and Institut fiir Strahlenbiologie, Technische Universitiit, Hannover, W. Germany
Summary Measurements of UV absorption by different parts of living Hep/2 cells have been obtained using a microspectrophotometer employing a low intensity of UV light. Between 255 nm and 280 nm, the nucleolus, nuclear sap and cytoplasm show significantly different absorptions. Living cells and cells fixed by freeze substitution show a similar pattern of UV absorption, although the absolute values may differ slightly.
The purpose of this work was to investigate the absorption of ultraviolet (UV) light by the nucleolus, nuclear sap, and cytoplasm of mammalian cells in tissue culture. This information is necessary to relate the effect of UV radiation on living cells, to the doses ab-