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γ-Rays kill grasshopper primary spermatocytes in groups
Mutation Research, 151 (1985) 73-76 Elsevier
73
MTR 04059
7-Rays kill grasshopper primary spermatocytes in groups A . A . A 1 - T a w e e l 1, M . A . S h a w k i t 1 a n d D . P . F o x 2 I Biology and Agriculture Faculty, Nuclear Research Centre, P.O. Box 765, Baghdad (lraq), and -" Department of Genetics, Unwersitv of Aberdeen, 2 Tillydrone Avenue, Aberdeen (Great Britain) (Received 17 October 1984) (Revision received 18 January 1985) (Accepted 30 January 1985)
Summary Primary spermatocyte killing by y-rays was studied in the grasshopper Heteracris littoralis in which spermatogenic development occurs in cysts containing a maximum of 64 cells during the first meiotic division. Cell killing at this stage is not random and mainly involves the death of whole cysts. The dose-response curve for cell killing has complex kinetics with at least two components but lacks any shoulder at low doses, thus indicating no repair of the lethal damage. Cell loss is apparent from surviving cysts as early as 45 min post irradiation but loss of > 24 cells is incompatible with cyst survival. Loss of fewer than 24 cells also is not random since certain values for cell loss are frequently observed while other, interspersed values are not seen at all.
Cell killing by X-rays usually occurs independently within a population. However, in beetle spermatogenesis A1-Taweel and Fox (1982) showed that primary spermatocytes (which are encysted and joined to each other by gap junctions within the cyst) do not die independently. Rather, cells may be lost from the cyst up to a maximum of 18 in species with 64 cells per cyst (28%) or 41 in species with 128 cells per cyst (32%) before the whole cyst dies. In addition, the lack of a shoulder on the dose-response curve for primary spermatocyte killing indicates a lack of repair of the lethal damage. In this paper it is shown that the primary spermatocytes of the grasshopper Heteracris littoralis are also killed in groups by y-rays.
Address for correspondence: Dr. D.P. Fox, Department of Genetics, University of Aberdeen, 2 Tillydrone Avenue, Aberdeen AB2 2TN, Great Britain.
Materials and methods Heteracris littoralis is a grasshopper with 2n = 23 ( ~ ) which is widely distributed in Iraq (Uvarov, 1938). It contains a maximum of 64 cells per primary spermatocyte cyst (AI-Taweel, unpublished work). Young adult males were collected in the field and maintained in the laboratory at 27 __+ I°C. Young adult males were exposed to different doses of y-radiation from a 6°Co y-ray source at a dose rate of 0.3-0.4 Gy/sec. Testes were fixed in alcohol-acetic acid (3:1 v / v ) for 12 h and then stained by the Feulgen reaction. Slide preparations were made by gently squashing individual testis follicles so as to maintain relative cell positions as far as possible on the slide. Slide preparations were made permanent by freezing with dry ice or liquid nitrogen. Under these conditions cell number per cyst can be determined for metaphase I cells which are easily recognised.
0027-5107/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
74
Results and discussion 0"2-
(i) Metaphase I o'st frequency following a single exposure to 10 Gy Table 1 and Fig. 1 show the change in frequency of primary spermatocyte cysts arriving at metaphase I at various time intervals up to 10 days post irradiation (p.i.). The cell numbers in each cyst were also determined. Cyst frequency declines rapidly to a maximum at 6 h p.i. which is 50% the preirradiation value. Over the next 3 days it recovers to the preirradiation level which is then maintained to 10 days p.i. In contrast, mean cell number per cyst declines rapidly to a minimum of about 50 cells per cyst (14 cells lost) by 1.5 h p.i. Even at 0.75 h p.i. cell loss from the cysts is quite clear. Thus, some cells are lost from individual cysts prior to any killing of the cysts themselves. Of course, some of the variation in cyst frequency could be due to delay in the cysts reaching metaphase I and not to cell killing, as meiotic delay was not determined. What does seem remarkable is the constancy of cell number per cyst throughout the variations in cyst frequency.
TABLE l FREQUENCY OF METAPHASE I CYSTS A N D MEAN CELL NUMBER IN THOSE CYSTS FOR PRIMARY SPERMATOCYTES PREVIOUSLY IRRADIATED WITH 10 Gy y-RAYS Time (h) post irradiation
Number of follicles scored ~
Met. 1 cysts/ follicle
Mean cell number at Met. 1
- 1 0.75 1.5 6 24 48 72 96 120 144 168 216 240
277 213 208 194 226 186 206 195 137 190 194 197 197
0.18 0.19 0.23 0.09 0.14 0.17 0.17 0.23 0.18 0.23 0.16 0.14 0.20
62.78 59.14 52.71 48.72 49.68 50.84 51.50 48.58 48.13 52.07 52.47 49.57 47.95
3 animals were used at each time interval except for the preirradiation sample (4).
-q
/
~0~ o
C>
-
-
-
0 o
o
--°~o
o
r s° /
L4o TIME
(DAYS)
AFTER
~RRADIATION
Fig. 1. Change in frequency of primary spermatocyte cysts ( , ) and cell number in those cysts (©) at intervals after exposure to 10 Gy y-rays.
(ii) Dose-response kinetics for primary spermatocyte killing by y-rays Table 2 and Fig. 2 show the change in frequency of primary spermatocyte cysts and cell number in those cysts which arrive at metaphase I 3 days p.i. with 0-30 Gy. It can be seen quite clearly that total cell killing is predominantly due to the killing of whole cysts, not of individual cells within those cysts. Cell number declines asymptotically to a mean of about 42 cells/cyst (22 cells lost) and this situation is illustrated in Fig. 3. Both maximum and minimum cyst values decline progressively to the 4 Gy dose but at higher dose levels the maxi-
TABLE 2 FREQUENCY OF METAPHASE I CYSTS AND MEAN CELL NUMBER IN THOSE CYSTS FOR PRIMARY SPERMATOCYTES IRRADIATED 3 DAYS PREVIOUSLY WITH 0-30 Gy "~,-RAYS y-Ray dose (Gy)
Number of follicles scored a
Met. I cysts/ follicle
Mean cell number at Met. I
0 1 2 4 6 8 10 15 30
277 242 259 241 256 263 240 431 471
0.18 0.16 0.13 0.14 0.11 0.11 0.10 0.04 0.03
62.78 53.98 49.03 47.06 48.19 46.86 44.97 42.69 42.09
a
4 animals were used at each irradiation except 6 Gy (3). 15 Gy (7) and 30 Gy (8).
> u~ J J
75
I o.olt~ ~..~__
O -0.4d Z !
.
~_l_
__!3o
° ~°~o'~' ,/
-o.0i O
i _i.0 ~ 0
r.,
~ -~o~ v I0
2'0
7 30
DOSE Gy
Fig. 2. Dose-response curve for killing of primary spermatocytes by y-rays. ×, mean surviving fraction of cells per cyst; e, mean surviving fraction of cysts per follicle; ©. mean surviving fraction of total primary spermatocytes.
'
dld
, 30
mum continues to decline while the minimum remains constant. Surviving cysts never had fewer than 40 cells. There is no hint of a shoulder in the survival curve for cells or cysts, indicating that there is no repair of the lethal damage. Also, the curve is complex in shape, having probably two components, at least one being non-exponential. If cell loss from cysts with a fixed cell number were a random process then distribution of cells loss would follow the Poisson distribution with mean cell loss being numerically equal to the
TABLE 3 MEAN AND VARIANCE OF CELL LOSS FROM PRIMARY SPERMATOCYTES IRRADIATED WITH 0-30 Gy y-RAYS ),-Ray dose (Gy)
Mean cell loss
Variance of cell loss
0 1 2 4 6 8 10 15 30
8.29 8.21 6.48 10.82 10.32 10.38 12.33 16.06 14.62
30.77 25.64 17.18 54.82 53.34 37.55 17.97 24.33 23.42
L ,
11
o
40 50 CELLS / CYST
60
70
Fig. 3. Frequency distribution of surviving primary spermatocytes in metaphase I cysts after exposure to 0-30 Gy 3,-rays.
variance of cell loss. Table 3 shows clearly that this is not so even for spontaneous cell loss in the control. In all cases after irradiation, even the highest dose levels compatible with some cyst survival and where minimum cell loss is still declining while maximum cell loss is static, the variance greatly exceeds the mean. Since cell loss from the cysts is non-random we may expect that cell loss will be mainly in groups of a specific size determined in some way by the structure of the spermatocyte cyst. In Fig. 4 the cell numbers in cysts from all dose levels have been pooled into a single histogram. This shows clearly that certain values for cell loss occur frequently, with 3, 4, 6, 9,
3
u- 1
30
40
50 CELLS/CYST
60
70
Fig. 4. Frequency distribution of surviving primary spermatocytes in metaphase I cysts pooled over all radiation doses.
76 14 and 16 being p a r t i c u l a r l y p r o m i n e n t , while 1, 5, 12, 15 and 17 were not recorded. S p e r m a t o c y t e killing by X-rays has previously been noted to affect p r i n c i p a l l y whole cysts in both Drosophila melanogaster a n d Pediculus corporis (Pontecorvo, 1944) a n d several species of Dermestes (A1-Taweel and Fox, 1982) b u t there do not a p p e a r to be c o m p a r a b l e d a t a for p r i m a r y sperm a t o c y t e killing in the m a m m a l i a n testis. However, there is some evidence that chains of differentiating s p e r m a t o g o n i a survive or die synchronously in the mouse ( H u c k i n s a n d Oakberg, 1978), a p h e n o m e n o n which also occurs in the i r r a d i a t e d beetle testis (AI-Taweel a n d Fox, 1982). F u r t h e r studies are now required with diverse a n i m a l
species in o r d e r to test for the generality of this p a t t e r n of cell killing by 7- or X-rays in synchronous groups of p r i m a r y spermatocytes.
References AI-Taweel, A.A., and D.P. Fox (1982) X-Ray induced cell death in the testis of dermestid beetles (Dermestes: Coleptera), Mutation Res., 106, 55-71. Huckins, C., and E.F. Oakberg (1978) Morphological and quantitative analysis of spermatogonia in mouse testes using whole mounted seminiferous tubules, lI. The irradiated testis, Anat. Rec., 192, 529-542. Pontecorvo, G. (1944) Synchronous mitoses and differentiation sheltering the germ track, Drosophila Inf. Serv., 18, 54-55. Uvarov, B.P. (1938) Orthoptera from Iraq and Iran, Zool. Ser. Field Museum Natur. Hist., 20, 439 451.
73
MTR 04059
7-Rays kill grasshopper primary spermatocytes in groups A . A . A 1 - T a w e e l 1, M . A . S h a w k i t 1 a n d D . P . F o x 2 I Biology and Agriculture Faculty, Nuclear Research Centre, P.O. Box 765, Baghdad (lraq), and -" Department of Genetics, Unwersitv of Aberdeen, 2 Tillydrone Avenue, Aberdeen (Great Britain) (Received 17 October 1984) (Revision received 18 January 1985) (Accepted 30 January 1985)
Summary Primary spermatocyte killing by y-rays was studied in the grasshopper Heteracris littoralis in which spermatogenic development occurs in cysts containing a maximum of 64 cells during the first meiotic division. Cell killing at this stage is not random and mainly involves the death of whole cysts. The dose-response curve for cell killing has complex kinetics with at least two components but lacks any shoulder at low doses, thus indicating no repair of the lethal damage. Cell loss is apparent from surviving cysts as early as 45 min post irradiation but loss of > 24 cells is incompatible with cyst survival. Loss of fewer than 24 cells also is not random since certain values for cell loss are frequently observed while other, interspersed values are not seen at all.
Cell killing by X-rays usually occurs independently within a population. However, in beetle spermatogenesis A1-Taweel and Fox (1982) showed that primary spermatocytes (which are encysted and joined to each other by gap junctions within the cyst) do not die independently. Rather, cells may be lost from the cyst up to a maximum of 18 in species with 64 cells per cyst (28%) or 41 in species with 128 cells per cyst (32%) before the whole cyst dies. In addition, the lack of a shoulder on the dose-response curve for primary spermatocyte killing indicates a lack of repair of the lethal damage. In this paper it is shown that the primary spermatocytes of the grasshopper Heteracris littoralis are also killed in groups by y-rays.
Address for correspondence: Dr. D.P. Fox, Department of Genetics, University of Aberdeen, 2 Tillydrone Avenue, Aberdeen AB2 2TN, Great Britain.
Materials and methods Heteracris littoralis is a grasshopper with 2n = 23 ( ~ ) which is widely distributed in Iraq (Uvarov, 1938). It contains a maximum of 64 cells per primary spermatocyte cyst (AI-Taweel, unpublished work). Young adult males were collected in the field and maintained in the laboratory at 27 __+ I°C. Young adult males were exposed to different doses of y-radiation from a 6°Co y-ray source at a dose rate of 0.3-0.4 Gy/sec. Testes were fixed in alcohol-acetic acid (3:1 v / v ) for 12 h and then stained by the Feulgen reaction. Slide preparations were made by gently squashing individual testis follicles so as to maintain relative cell positions as far as possible on the slide. Slide preparations were made permanent by freezing with dry ice or liquid nitrogen. Under these conditions cell number per cyst can be determined for metaphase I cells which are easily recognised.
0027-5107/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
74
Results and discussion 0"2-
(i) Metaphase I o'st frequency following a single exposure to 10 Gy Table 1 and Fig. 1 show the change in frequency of primary spermatocyte cysts arriving at metaphase I at various time intervals up to 10 days post irradiation (p.i.). The cell numbers in each cyst were also determined. Cyst frequency declines rapidly to a maximum at 6 h p.i. which is 50% the preirradiation value. Over the next 3 days it recovers to the preirradiation level which is then maintained to 10 days p.i. In contrast, mean cell number per cyst declines rapidly to a minimum of about 50 cells per cyst (14 cells lost) by 1.5 h p.i. Even at 0.75 h p.i. cell loss from the cysts is quite clear. Thus, some cells are lost from individual cysts prior to any killing of the cysts themselves. Of course, some of the variation in cyst frequency could be due to delay in the cysts reaching metaphase I and not to cell killing, as meiotic delay was not determined. What does seem remarkable is the constancy of cell number per cyst throughout the variations in cyst frequency.
TABLE l FREQUENCY OF METAPHASE I CYSTS A N D MEAN CELL NUMBER IN THOSE CYSTS FOR PRIMARY SPERMATOCYTES PREVIOUSLY IRRADIATED WITH 10 Gy y-RAYS Time (h) post irradiation
Number of follicles scored ~
Met. 1 cysts/ follicle
Mean cell number at Met. 1
- 1 0.75 1.5 6 24 48 72 96 120 144 168 216 240
277 213 208 194 226 186 206 195 137 190 194 197 197
0.18 0.19 0.23 0.09 0.14 0.17 0.17 0.23 0.18 0.23 0.16 0.14 0.20
62.78 59.14 52.71 48.72 49.68 50.84 51.50 48.58 48.13 52.07 52.47 49.57 47.95
3 animals were used at each time interval except for the preirradiation sample (4).
-q
/
~0~ o
C>
-
-
-
0 o
o
--°~o
o
r s° /
L4o TIME
(DAYS)
AFTER
~RRADIATION
Fig. 1. Change in frequency of primary spermatocyte cysts ( , ) and cell number in those cysts (©) at intervals after exposure to 10 Gy y-rays.
(ii) Dose-response kinetics for primary spermatocyte killing by y-rays Table 2 and Fig. 2 show the change in frequency of primary spermatocyte cysts and cell number in those cysts which arrive at metaphase I 3 days p.i. with 0-30 Gy. It can be seen quite clearly that total cell killing is predominantly due to the killing of whole cysts, not of individual cells within those cysts. Cell number declines asymptotically to a mean of about 42 cells/cyst (22 cells lost) and this situation is illustrated in Fig. 3. Both maximum and minimum cyst values decline progressively to the 4 Gy dose but at higher dose levels the maxi-
TABLE 2 FREQUENCY OF METAPHASE I CYSTS AND MEAN CELL NUMBER IN THOSE CYSTS FOR PRIMARY SPERMATOCYTES IRRADIATED 3 DAYS PREVIOUSLY WITH 0-30 Gy "~,-RAYS y-Ray dose (Gy)
Number of follicles scored a
Met. I cysts/ follicle
Mean cell number at Met. I
0 1 2 4 6 8 10 15 30
277 242 259 241 256 263 240 431 471
0.18 0.16 0.13 0.14 0.11 0.11 0.10 0.04 0.03
62.78 53.98 49.03 47.06 48.19 46.86 44.97 42.69 42.09
a
4 animals were used at each irradiation except 6 Gy (3). 15 Gy (7) and 30 Gy (8).
> u~ J J
75
I o.olt~ ~..~__
O -0.4d Z !
.
~_l_
__!3o
° ~°~o'~' ,/
-o.0i O
i _i.0 ~ 0
r.,
~ -~o~ v I0
2'0
7 30
DOSE Gy
Fig. 2. Dose-response curve for killing of primary spermatocytes by y-rays. ×, mean surviving fraction of cells per cyst; e, mean surviving fraction of cysts per follicle; ©. mean surviving fraction of total primary spermatocytes.
'
dld
, 30
mum continues to decline while the minimum remains constant. Surviving cysts never had fewer than 40 cells. There is no hint of a shoulder in the survival curve for cells or cysts, indicating that there is no repair of the lethal damage. Also, the curve is complex in shape, having probably two components, at least one being non-exponential. If cell loss from cysts with a fixed cell number were a random process then distribution of cells loss would follow the Poisson distribution with mean cell loss being numerically equal to the
TABLE 3 MEAN AND VARIANCE OF CELL LOSS FROM PRIMARY SPERMATOCYTES IRRADIATED WITH 0-30 Gy y-RAYS ),-Ray dose (Gy)
Mean cell loss
Variance of cell loss
0 1 2 4 6 8 10 15 30
8.29 8.21 6.48 10.82 10.32 10.38 12.33 16.06 14.62
30.77 25.64 17.18 54.82 53.34 37.55 17.97 24.33 23.42
L ,
11
o
40 50 CELLS / CYST
60
70
Fig. 3. Frequency distribution of surviving primary spermatocytes in metaphase I cysts after exposure to 0-30 Gy 3,-rays.
variance of cell loss. Table 3 shows clearly that this is not so even for spontaneous cell loss in the control. In all cases after irradiation, even the highest dose levels compatible with some cyst survival and where minimum cell loss is still declining while maximum cell loss is static, the variance greatly exceeds the mean. Since cell loss from the cysts is non-random we may expect that cell loss will be mainly in groups of a specific size determined in some way by the structure of the spermatocyte cyst. In Fig. 4 the cell numbers in cysts from all dose levels have been pooled into a single histogram. This shows clearly that certain values for cell loss occur frequently, with 3, 4, 6, 9,
3
u- 1
30
40
50 CELLS/CYST
60
70
Fig. 4. Frequency distribution of surviving primary spermatocytes in metaphase I cysts pooled over all radiation doses.
76 14 and 16 being p a r t i c u l a r l y p r o m i n e n t , while 1, 5, 12, 15 and 17 were not recorded. S p e r m a t o c y t e killing by X-rays has previously been noted to affect p r i n c i p a l l y whole cysts in both Drosophila melanogaster a n d Pediculus corporis (Pontecorvo, 1944) a n d several species of Dermestes (A1-Taweel and Fox, 1982) b u t there do not a p p e a r to be c o m p a r a b l e d a t a for p r i m a r y sperm a t o c y t e killing in the m a m m a l i a n testis. However, there is some evidence that chains of differentiating s p e r m a t o g o n i a survive or die synchronously in the mouse ( H u c k i n s a n d Oakberg, 1978), a p h e n o m e n o n which also occurs in the i r r a d i a t e d beetle testis (AI-Taweel a n d Fox, 1982). F u r t h e r studies are now required with diverse a n i m a l
species in o r d e r to test for the generality of this p a t t e r n of cell killing by 7- or X-rays in synchronous groups of p r i m a r y spermatocytes.
References AI-Taweel, A.A., and D.P. Fox (1982) X-Ray induced cell death in the testis of dermestid beetles (Dermestes: Coleptera), Mutation Res., 106, 55-71. Huckins, C., and E.F. Oakberg (1978) Morphological and quantitative analysis of spermatogonia in mouse testes using whole mounted seminiferous tubules, lI. The irradiated testis, Anat. Rec., 192, 529-542. Pontecorvo, G. (1944) Synchronous mitoses and differentiation sheltering the germ track, Drosophila Inf. Serv., 18, 54-55. Uvarov, B.P. (1938) Orthoptera from Iraq and Iran, Zool. Ser. Field Museum Natur. Hist., 20, 439 451.