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Unfertilized rabbit ova in P32-autoradiography
574
BRIEF
UNFERTILIZED
RABBIT
OVA IN P=AUTORADIOGRAPHY
ERIK Women’s clinic,
Karolinska
NOTE
ODEBLAD
Institutet,
Sabbatsberg Hospital,
Received January
MATERIAL
AND
Stockholm,
Sweden
24, 1951
METHODS
IO adult female rabbits have been used. 2 were anoestric and untreated, 6 were anoestric and treated with human chorionic gonadotrophin (Pregnyl), as shown in table I, and 2 were pregnant. Two hours before death each rabbit received 1.2-6 m C Intravenous injection of barbiturate was of P32 intravenously as orthophosphate. used to kill the rabbits. Immediately after death the ovaries were fixed in a solution of 6 per cent formalin+l/, per cent trichloracetic acid in distilled water, the first two hours at +4” C, then at + 19” C for 22 hours. The ovaries were frozen-sectioned in IO p sections, mounted on glass slides, passed through 3 baths of neutral distilled water for 1/2 hour, dried, and passed through 3 baths equal parts of ethanol and ether for 1/2 hour. They were then dried and subjected to autoradiography. Gevaert noscreen X-ray film or Gevaert Dentus Rapid film was pressed directly against the section for 4-20 days in iron presses. After this exposure, development, with Gevaert G 209 A, was performed for 4 minutes at + 19” C. The resolution was approximately 25 IL. Control exposures With non-radioactive material gave no blackening of the film. About 150 autoradiographs and the corresponding sections, stained with haemaluneosin, were examined. Follicles having an apparent diameter of 500-l 000 p, showing no signs of atresia, and containing centrally cut ova (with visible nucleolus), having an apparent diameter of 150-200 EL,were selected for study. It was possible to find 16 such subjects. The number of grains (N) was counted in oil immersion at 1 700 times of magnification, using eye-piece squared micrometer, each square corresponding to 37 p2. The number of grains per 37 p2 was counted in the central parts of the ova (No), in the adjacent granulosa cells (Ns) in the adjacent part of hollow space of the follicle (antrum folliculi) (Na), if visible, and in the fog region (Nf). The ratios z
(I) and E t3
(II) Is
were calculated and the statistical errors considered. Thus the exposure from the ovum was compared with that from the granulosa tissue (I) or the exposure from the antrum folliculi was compared with that from the granulosa tissue (I). The ex-
Unfertilized
rabbit ova in P32-Auforadiography
575
enlarged 20 times, showing a follicle of a size of 900 p, containing an Fig. 1. Autoradiograph, ovum of ibout 150 p size. The isotope distribution in the ovum, antrum and granulosa areas are clearly visible. TABLE
Rabbit nr 1
Hormonal
Ratio II
0.32 i- 0.09
0.24 IL 0.09
0.08&0.14
0.25 + 0.08
0.16t0.13
0.23 F 0.07
0.17+0.11
0.22 i 0.08
0.11f0.11
Untreated,
not pregnant
-
-
Ratio 1
treatment
-
I
L
Ratio IRatio II
0.35-to.10 n
2 3
Pregnyll
Pregnyl
Pregnyl
10
0.50 * 0.11 0.41 IL 0.08 0.33 f 0.08
28 and 3 days earlier
0.42f0.12
Untreated,
pregnant
7 days
Untreated,
pregnant.
20 days
T Mean .L 1 Each
0.35 f 0.09 0.41 k 0.09
15 days earlier
0
8 9
0.51* 0.08
0
6 7
0.41 f 0.10 0.33kO.11
5 days earlier P
4 5
0.40 IL 0.10
dosis of Pregnyl
0.33 + 0.08 0.39 t 0.09 0.40 k 0.09
-
0.30 * 0.09
0.24 + 0.08
0.39 + 0.04
0.24 f 0.05
L was 300 IU human
chorionic
gonadotrophin.
0.0610.13
T -
0.15kO.06
576
E. Odeblad
posure from the ovum was also compared with that of the antrum by subtraction of II from I. In the region of exposure used the number of grains is approximately proportional to the amount of P32 emitting the radiation. RESULTS
The results are shown in table I and examplified in fig. 1. The amount of P32 per unit tissue volume seems less within the ovum than within the granulosa tissue, but seems somewhat more than in the follicular fluid. No appreciable differences are seen between anoestric, hormone treated or pregnant rabbits. DISCUSSION
The histologic treatment has with great probability dissolved much of the acid-soluble and lipid-soluble phosphorus. The remaining phosphorus in the section, therefore, must to a relatively great extent belong to nucleic acids and proteins. Because of the dependence of exposure and resolution on the spatial distribution of isotope within the histologic section the results do not directly indicate the true amounts of P 32 in the remaining fraction in the corresponding areas, but form a rough guide to the presence of P32 in different structures. It is possible to criticize the calculation of the ratios because the number of granulosa cells varies from case to case. Due to similar degrees of follicular development, this variation is, however, slight. This calculation has the avantage, that there is no appreciable dependence on the section thickness, dosage of isotope, length of exposure or time of development. The findings are of interest with regard to the results of Lindberg (1, 2) and Villie, Lowens and Gordon (3) that unfertilized ova, contrary to fertilized ova, do not to any appreciable extent permit the penetration of labelled orthophosphate. SUMMARY
By means of autoradiography, permitting a resolution of about 25 p, it is indicated that acid- and lipid-insoluble P32 is present only in small amounts in unfertilized, nearly mature rabbit ova. No changes are indicated in treatment with human chorionic gonadotrophin or in pregnancy. The author is indebted to the Pharmacia Co for having placed the gonadotrophic preparation Pregnyl at his disposal. LITERATURE 1. LINDBERG,
2. __ 3. VILLIE,
ExpfI.
O., Arkiu. Kemi, 26 B (1948). Cell Res., 1, 105 (1950).
C. A.,
LOWENS,
M., & GORDON,
M., J. Cellular
Comp. Physiol.,
33, 93 (19491.
BRIEF
UNFERTILIZED
RABBIT
OVA IN P=AUTORADIOGRAPHY
ERIK Women’s clinic,
Karolinska
NOTE
ODEBLAD
Institutet,
Sabbatsberg Hospital,
Received January
MATERIAL
AND
Stockholm,
Sweden
24, 1951
METHODS
IO adult female rabbits have been used. 2 were anoestric and untreated, 6 were anoestric and treated with human chorionic gonadotrophin (Pregnyl), as shown in table I, and 2 were pregnant. Two hours before death each rabbit received 1.2-6 m C Intravenous injection of barbiturate was of P32 intravenously as orthophosphate. used to kill the rabbits. Immediately after death the ovaries were fixed in a solution of 6 per cent formalin+l/, per cent trichloracetic acid in distilled water, the first two hours at +4” C, then at + 19” C for 22 hours. The ovaries were frozen-sectioned in IO p sections, mounted on glass slides, passed through 3 baths of neutral distilled water for 1/2 hour, dried, and passed through 3 baths equal parts of ethanol and ether for 1/2 hour. They were then dried and subjected to autoradiography. Gevaert noscreen X-ray film or Gevaert Dentus Rapid film was pressed directly against the section for 4-20 days in iron presses. After this exposure, development, with Gevaert G 209 A, was performed for 4 minutes at + 19” C. The resolution was approximately 25 IL. Control exposures With non-radioactive material gave no blackening of the film. About 150 autoradiographs and the corresponding sections, stained with haemaluneosin, were examined. Follicles having an apparent diameter of 500-l 000 p, showing no signs of atresia, and containing centrally cut ova (with visible nucleolus), having an apparent diameter of 150-200 EL,were selected for study. It was possible to find 16 such subjects. The number of grains (N) was counted in oil immersion at 1 700 times of magnification, using eye-piece squared micrometer, each square corresponding to 37 p2. The number of grains per 37 p2 was counted in the central parts of the ova (No), in the adjacent granulosa cells (Ns) in the adjacent part of hollow space of the follicle (antrum folliculi) (Na), if visible, and in the fog region (Nf). The ratios z
(I) and E t3
(II) Is
were calculated and the statistical errors considered. Thus the exposure from the ovum was compared with that from the granulosa tissue (I) or the exposure from the antrum folliculi was compared with that from the granulosa tissue (I). The ex-
Unfertilized
rabbit ova in P32-Auforadiography
575
enlarged 20 times, showing a follicle of a size of 900 p, containing an Fig. 1. Autoradiograph, ovum of ibout 150 p size. The isotope distribution in the ovum, antrum and granulosa areas are clearly visible. TABLE
Rabbit nr 1
Hormonal
Ratio II
0.32 i- 0.09
0.24 IL 0.09
0.08&0.14
0.25 + 0.08
0.16t0.13
0.23 F 0.07
0.17+0.11
0.22 i 0.08
0.11f0.11
Untreated,
not pregnant
-
-
Ratio 1
treatment
-
I
L
Ratio IRatio II
0.35-to.10 n
2 3
Pregnyll
Pregnyl
Pregnyl
10
0.50 * 0.11 0.41 IL 0.08 0.33 f 0.08
28 and 3 days earlier
0.42f0.12
Untreated,
pregnant
7 days
Untreated,
pregnant.
20 days
T Mean .L 1 Each
0.35 f 0.09 0.41 k 0.09
15 days earlier
0
8 9
0.51* 0.08
0
6 7
0.41 f 0.10 0.33kO.11
5 days earlier P
4 5
0.40 IL 0.10
dosis of Pregnyl
0.33 + 0.08 0.39 t 0.09 0.40 k 0.09
-
0.30 * 0.09
0.24 + 0.08
0.39 + 0.04
0.24 f 0.05
L was 300 IU human
chorionic
gonadotrophin.
0.0610.13
T -
0.15kO.06
576
E. Odeblad
posure from the ovum was also compared with that of the antrum by subtraction of II from I. In the region of exposure used the number of grains is approximately proportional to the amount of P32 emitting the radiation. RESULTS
The results are shown in table I and examplified in fig. 1. The amount of P32 per unit tissue volume seems less within the ovum than within the granulosa tissue, but seems somewhat more than in the follicular fluid. No appreciable differences are seen between anoestric, hormone treated or pregnant rabbits. DISCUSSION
The histologic treatment has with great probability dissolved much of the acid-soluble and lipid-soluble phosphorus. The remaining phosphorus in the section, therefore, must to a relatively great extent belong to nucleic acids and proteins. Because of the dependence of exposure and resolution on the spatial distribution of isotope within the histologic section the results do not directly indicate the true amounts of P 32 in the remaining fraction in the corresponding areas, but form a rough guide to the presence of P32 in different structures. It is possible to criticize the calculation of the ratios because the number of granulosa cells varies from case to case. Due to similar degrees of follicular development, this variation is, however, slight. This calculation has the avantage, that there is no appreciable dependence on the section thickness, dosage of isotope, length of exposure or time of development. The findings are of interest with regard to the results of Lindberg (1, 2) and Villie, Lowens and Gordon (3) that unfertilized ova, contrary to fertilized ova, do not to any appreciable extent permit the penetration of labelled orthophosphate. SUMMARY
By means of autoradiography, permitting a resolution of about 25 p, it is indicated that acid- and lipid-insoluble P32 is present only in small amounts in unfertilized, nearly mature rabbit ova. No changes are indicated in treatment with human chorionic gonadotrophin or in pregnancy. The author is indebted to the Pharmacia Co for having placed the gonadotrophic preparation Pregnyl at his disposal. LITERATURE 1. LINDBERG,
2. __ 3. VILLIE,
ExpfI.
O., Arkiu. Kemi, 26 B (1948). Cell Res., 1, 105 (1950).
C. A.,
LOWENS,
M., & GORDON,
M., J. Cellular
Comp. Physiol.,
33, 93 (19491.