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Effects of radiocobalt irradiation of rabbit spermatozoa in vitro on fertilization and early development.

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EFFECTS O F RADIOCOBALT IRRADIATION
O F RABBIT SPERMATOZOA I N VITRO
ON FERTILIZATION AND EARLY
DEVELOPMENT
M. C. CHANG, DOROTHY M. HUNT AND E.B.ROMANOFF
Worchester Foundation for Experimental Biology, Shrewsbury, and
Department of Biology, Boston University, Boston, Massachusetts
EIQHTEEN FIQUBES
A research project supported by the Atomic Energy Commission was undertaken in this laboratory to investigate the
effects of irradiation on mammalian ova in vitro. I n order
to develop needed techniques and to comprehend the effect of
radiation on germ cells in general it was thought profitable
to first study the effect of radiocobalt irradiation on spermatozoa in vitro.
I t is known that during spermatogenesis the male germ cells
are extremely sensitive to irradiation while the mature spermatozoa are extremely resistant to such exposure. X-irradiated spermatozoa may retain both their motility and fertilizing capacity but are unable to initiate normal development
of the fertilized ova due to latent damage to the chromatin.
I n their extensive study of the effects on embryonic development of X-irradiation of rabbit spermatozoa in z d r o , Amoroso
and Parkes ('47) concluded that exposure of spermatozoa to
50 r and 100 r did not interfere with fertilization, segmentation
of the ovum, or embryonic and fetal development ; following
irradiation with 500 r, litter size was reduced and with 1,000 r
no implantation "as observed; with 2,500 r all tuba1 ova
This study was supported by the Atomic Energy Commission under Contract
AT (30-1)-1943. Thanks are due to Dr. G. Pincus for constant interest in this
study and t o Mrs. P. Goff for looking a f tr r the animals.
211
212
M. C. CHANG, D. M. H U N T A N D
E. B. ROMANOPF
showed lack or arrest of development. They concluded further
that irradiated spermatozoa had penetrated the ova at all dose
levels (50 to 100,000 r ) but the process was often delayed;
polyspermy was not uncommon. With the higher doses the
male pronucleus was abnormal, syngamy was delayed and
irregular, and many activated ova failed t o divide ; the arrest
of segmentation was associated with the occurrence of irregular nuclei and anucleate or multinucleate cells. Since most
of their ova were examined 40 hours after ovulation it was
thought that the nuclear behavior during fertilization and
cleavage should be re-investigated.
The present study deals with ova examined 8, 38 and
132 hours after ovulation to investigate the effect of irradiation on the sequence of fertilization and of early degeneration.
Examination at late pregnancy was also made to determine
the possibility of the abnormality of young. It was also
thought to be of interest to compare the effects of gammairradiation with X-irradiation.
METHODS AND PROCEDURES
Adult female rabbits of mixed breeds (mainly New Zealand
white) purchased from local farms were kept in separate cages
in the laboratory for about 3 weeks before use. Semen from
fertile males reared in the laboratory was collected by means
of an artificial vagina (Macirone and Walton, '38). Two or
three ejaculates of different males mere mixed and added with
about an equal volume of 0.9% NaCl in order to increase the
volume for irradiation and insemination. The semen samples,
about 1 to 3 ml in short glass tubes, were attached at various
distances from the source of the radiocobalt for a certain
length of time in order t o achieve the dose of irradiation
required. The dosage was ascertained by actual measurement
of ionization of air with a Victoreen r-Meter by Mr. R. L.
Bersin of Tracerlab, Boston. Immediately after irradiation, a
semen sample of 0.5ml was artificially inseminated into the
vagina of a female rabbit (Walton, '30) and sheep pituitary
extract preparation was administered intravenously for the
IRRADIATION O F RABBIT SPERMATOZOA
213
induction of ovulation. The number of spermatozoa in the
sample ~7asestimated from haemocytometer counts and the
number of sperm inseminated to each rabbit was calculated.
I n general about 4 females were inseminated at a time.
About 18 or 48 hours after insemination the left Fallopian
tube of a female was removed and the ova were recovered by
flushing the tube with saline. At either 6 days or 2 6 2 5 days
after insemination, the animals were sacrificed and the gravid
uterus was flushed with saline for the recovery of blastocysts
or cut open to examine the conceptuses.
The ova recovered from the tube were mounted in saline on a
slide, fixed and stained according to the description of Chang
( '52; '55a). The ova were examined or photographed under a
phase-contrast microscope before fixation, after fixation with
acetic acid, and after staining either with lacmoid (Darlington
and LaCour, '39) for chromosomes or with methyl green
and pyronine (Jordan and Baker, '55) for pronucleus. Blastocysts recovered 6 days after insernination were either mounted
and stained, as in the case of tuba1 ova, or fixed, spread and
stained according to Feulgen method as described elsewhere
(Chang, '54).
RESULTS
Motility and number of spermatozoa bweminated. The
motility of spermatozoa after irradiation with 90 to 6,500 r
was quite good, There was no striking difference as compared
with control samples. Following irradiation with 32,200 r, the
motility was poor; with 65,000 r, only a few spermatozoa
showed progressive movement under a 4 mm objective field.
It was noted further that no motility at all was observed in
a sample having a low concentration of sperm, e.g. 20 million
spermatozoa per ml, after irradiation with 65,000 r. Thus, very
concentrated semen, 45 to 125 million per rabbit, was inseminated after the irradiation with 32,200 or 65,00Or, and 30 to
75 million spermatozoa were inseminated after a lower dose
of irradiation. From the results of table 1,it can be seen that
the proportion of fertilized ova is roughly correlated with the
dosage of irradiation.
27
11
12
6
11
800
6,500
32,200
65,000
32
43
23
25
21
59
84
92
93
91
%
* 8 : Pronuclear
stage; 20 : 2 cells; 1 : 3 cells.
*All unfertilized ova come from sterile does.
Including unfertilized ova.
54
51
25
23
11
,No*
$,
Pronu.
clei
FERTILIZATION
HRS. AFTER
INSEX.)
(18
90
RABBITS
INBEM.
NO. OF
59'
15'
29'
31'
21
No.
,",'
,
29*
10
0
0
0
0
1
5
17
4
9
19
20
8+
eeb
1
5-6
cells
0
2-4
cells
( 2 DAY8 AFTER' INSEX.)
CLEAVAGE
0
0
5
55
95
%
norm.
38
41
36
2
ova
36
18
5
ova
Deg.
2
19
9
Deg.
blast.
0
4
22
36
25
0
43
4
0
0
28
fetus
Norm.
1
5
Deg.
yNo.
k r embr.
0
11
65
%
normal
EXBEYONIC DEVELQPM=NT
DAYS APTER INBEY.)
(24-25
10
61
%
2°K: norm.
BWSTOCYS'F DEVELOPMENT
( 6 DAYS AFTER INBEY.)
TABLE 1
Effect of radiboobatt irradiations of rabbit spermatozoa on fertiliaation and early development
IRRADIATION O F RABBIT SPERMATOZOA
215
Fertilization of ova by irradiated spermatozoa. Since it is
known that fertilization of ova is inhibited in pseudopregnant
rabbits (Murphree, et. al. '47)data were rejected whenever the
presence of corpora lutea in the ovary noticed. By the presence of first maturation spindle an unfertilized ovum can be
easily distinguished from a fertilized ovum which has either
pronuclei and polar bodies or several blastomeres. At 6 days
after insemination, however, an unfertilized ovum is not so
easy to distinguish from an early degenerated fertilized ovum.
As shown in table 1, following insemination with sperm
irradiated at 901-to 6,500r the rate of fertilization is not
different from that of normal mating. Following insemination
with sperm irradiated at 32,200r, 4 out of 6 animals had 96%
of fertilization (47 out of 49 eggs) while the other 2 animals
had 35% of fertilization (6 out of 17). This demonstrated a
tendency towards a decrease of fertilizing capacity in the
irradiated sperm with higher doses. When irradiation was at
65,000 r, 5 out of 11 rabbits had no ovum fertilized while the
other rabbits had 98% of fertilization (62 out of 63 ova). It
may be that the physiological condition of individual females
plays an important role in transportation of weak or irradiated spermatozoa and this may cause the decrease of fertilization rate. Since all the unfertilized ova had no sperm or only a
few (1 to 5 ) sperm on the zona pellucida and since the majority
of the fertilized ova had 5 to 20 sperm on the zona pellucida it
shows that following the irradiation with higher dose a main
factor for non-fertilization is the incapacity of sperm to reach
the site of fertilization rather than the incapacity of sperm to
fertilize the ova.
Prownuclei of fertilixed ova. When the ova were examined
18 hours after the insemination with irradiated sperm and
ovulation-inducing injection, that is, about 8 hours after
ovulation, most of the ova had two pronuclei very close to
each other in the center of the cytoplasm (figs. l a and l b )
which is a normal occurrence at this time. There is, however,
as presented in table 2, about 10% exception to this occurrence
(14 out of 144 ova) : four ova had their pronuclei at various
216
M.
c. CHANG, D.
M. HUNT AND
E. B. ROMANOFF
distances from each other, but their pronuclei are well formed
(figs. 2a and 2b). This is probably independent of the dose
used as shown in table 2. I n the normal sequence of rabbit
fertilization the male and female elements are separated from
each other only at about 2 to 4 hours after ovulation and their
pronuclei are not well formed at that time (Chang, ’50). Thus,
if the peripheral location of pronuclei is not a normal variation but an effect of irradiation of spermatozoa it seems that
it is due neither to the delay of irradiated sperm t o penetrate
the ova nor to the delay of pronuclear formation but is due t o a
lack of attraction to each other. Based on the facts that the
TABLE 2
Pronuclear behavior of ova fertilized by irradiated spermatozoa
NUMBER OF O V A
DOSE
(R)
Total
Pronuclei
apart
(delayed
Fragmen. Fragmentation
tation of
of female
male
aynganiy 1 ) pronuclrus pronucleus
90
21
1
800
6,500
32,200
66,000
25
23
43
1
32
144
2
4
Total
Three
pronuclei
(polyspermy) (
One
w
~
~
B
1
3
1
1
1
6
I ?
1
2
1
1
1
female pronucleus is expected to be near the polar bodies and
that it is smaller than male pronucleus, it was observed that
out of 144 ova, 6 had a fragmented female pronucleus (figs.
3a and 3b) and 2 had a fragmented male pronucleus (fig. 4)
following the fertilization of sperm irradiated with 800r to
32,200 r. It is uncertain, however, whether or not the fragmentation of the pronucleus is due to irradiation of sperm because
(a) a small fragment beside the pronucleus has been observed
occasionally at about 18 hours after normal mating, (b) the
proportion of ova with fragmented female pronucleus was
higher than that with male pronucleus and (c) the fragmentation does not seem to be related to the dose levels (table 2).
One ovum fertilized by sperm irradiated with 32,200 r had 3
pronuclei, one female and two male pronuclei (figs. 5a and 5b),
~
~
~
)
IFiBADIATION O F BABBIT SPERMATOZOA
217
demonstrating the possibility of polyspermy after irradiation
with higher doses. One ovum fertilized by sperm irradiated
with 65,000 r had only one female pronucleus (fig. 6) showing
the possibility of gynogenetic activation of this ovum.
Cleavage of the fertilized ova. Following insemination of
irradiated spermatozoa, the mechanism of cleavage was very
much injured as shown 48 hours after insemination though
the probability of fertilization was quite high at all dose levels.
From the results presented in table 1,it is quite clear that the
damage is in proportion to the dose applied to the sperm. I n
the normal sequence of the cleavage of rabbit ova, when
examined 48 hours after mating or after insemination, 16 to 32
celled ova are expected (Gregory, '30; Chang, '50). I n the
present study when examined 48 hours after insemination,
only a fern ova fertilized by sperm irradiated with the lowest
dosage, 90 r, had cleaved into 16 cells and most of them were
at the 8 celled stage (fig. 7 ) . Following irradiation of sperm
with 800 r, about half the number of the ova cleaved to 8 cells
stage but no ovum cleaved to 16 cells stage (table 1and fig. 8)
by this time. Irradiation with 6,500 r, only one ovum cleaved to
8 cells and most of them were at a 2 to 4 cell stage (fig. 9).
After irradiation with 32,200r or with 65,000r, no ovum
cleaved to more than 3 cells (fig. lo), and a few ova definitely
fertilized by sperm irradiated at 65,000r failed to cleave at
all (fig. 11).It is therefore quite clear that the rate of cleavage
and the mechanism of cleavage are injured by the irradiation
of sperm and that the higher the dose the sooner the damage
appears. Since most of the ova that failed to cleave further
following insemination with sperm irradiated at 65,000 r had
small dark granules showing degenerative changes in their
nuclei (fig. 12), and since a few ova had spindles and chromosomes at metaphase (fig. 13), it appears that the incapacity
of mitotic division may occur at any phase of mitosis.
Blastocyst formation. I n the normal development of fertilized rabbit ova, 6 days after mating o r insemination, blastocysts were found to be about 3 to 4 mm in diameter (Chang,
'50). Occasionally, however, a small blastocyst measured
218
M. C. CHANG, D. M. H U N T AND
E. B. ROMANOFF
about 2mm in diameter has been observed. I n the present
study, a blastocyst of 2mm diameter or greater, 6 days after
insemination, was arbitrarily considered as a normal blastocyst. Blastocysts either shrunk (separation of trophoblast
from zona pellucida) or with dark granulated cells and usually
smaller than 2 mm were considered degenerated.
The data in table 1 show that 61 and 10 per cent of normal
blastocysts were formed after the fertilization by sperm
irradiated with 90 r and with 800 r respectively (fig. 14) but
only two degenerated blastocysts out of 38 ova were recovered
(as illustrated in fig. 14) after the irradiation of sperm with
6,500 r. This again points to the fact that the higher dose of
irradiation leads to severe damage to cleavage of fertilized ova
at an early stage. Since the proportion of normally cleaved
ova is higher than that of blastocysts (95 vs. 61% at 90 r, 55 vs.
10% at 800 r, and 5 vs. 0% at 6,500 r ) it appears that the damage is revealed progressively from oleavage to blastocyst formation. As for the cytology of the blastocyst, the chromosomal
and nuclear configuration of the normal blastocyst are
apparently normal (fig. 15). In the degenerated blastocyst
more necrotic nuclei were observed but nuclei at various
phases of mitosis were also observed (fig.16).
Fetal development. m7hen the uterus was examined 24 to 25
days after insemination, the percentage of normal fetuses
formed in terms of the number of corpora lutea was 65 after
the irradiation of sperm with 90r. Since 74% of normal
fetuses resulted following the insemination of untreated sperm
and an operation involving the removal of one ovary (Chang,
'55b), it seems that the probability of normal fetal development is only slightly lower than with insemination of normal
sperm but the difference is not statistically significant. Considering the fact that the proportion of normal blastocysts and
the proportion of normal fetuses are similar (61vs. 65% at
90 r, 10 vs. ll%, at 800 r and 0 vs. 0% at 6,500 r ) , it is suggested that once a normal blastocyst is formed implantation
and normal development is not prevented. All the fetuses
examined were morphologically normal and their sex ratio
IRRADIATION OF RABBIT SPERMATOZOA
219
was not disturbed. The proportion of degeneration after
implantation was 8% (6 out of 79 corpora lutea) as shown by
the presence of placentae in the uterus. This is not due to the
irradiation of sperm because 11% of degeneration after
implantation has seen obtained after insemination with normal
sperm (Chang, '55b).
DISCUSSTON
The results of this study confirm in general most of the
findings described by Amoroso and Parkes ( '47). However,
there are a few points in our study which are in disagreement
with them.
So far as we could judge from our material the entry of
irradiated spermatozoa was not delayed as shown by the
well-formed pronuclei and the close contact of both nuclei at
8 hours after ovulation, although 3 to 10 hours of delay in
some instances was reported by them. The occurrence of a
proportion of polyspermic ova at all treatment levels between
1,000 r and 50,000 r has been reported by them but only one
probable polyspermic ovum fertilized by sperm irradiated at
32,200r was observed in the present study. I n their figures
3b and 3c, plate 3, the round dark spot is more like the
nucleolus of a pronucleus than a sperm head. From their
figure 10, plate 3, one wonders whether the two pronuclei above
the male pronucleus are fragmentation products of a female
pronucleus o r one male and one female pronucleus. I n their
figure 18a, plate 5, the presence of several spermatozoa in one
blastomere of a 7-celled ovum without change in their form is
very extraordinary and the attachment of spermatozoa at
preparation is quite possible. Since some of their inseminations
were done at 2 1/4hours before ovulation, the polyspermic ova,
if there were some, may be due to the fertilization of aged ova
as reported by Austin and Rraden ( '53).
Activated ova which fail to divide have been observed in our
study in only 8 out of 29 ova fertilized by sperm irradiated
at 65,000 r. However, these uncleaved ova all had well formed
pronuclei and a second polar body when examined 2 days after
220
M. C. CHANG, D. M. H U N T AND E. B. ROMANOFF
insemination (fig. 11). If activation of the ovum connotes
the extrusion of a second polar body and the inward migration
of the female elements, which is a general feature after sperm
penetration, we agree with them that spermatozoa exposed
t o 1,000 r to 100,000 r retain their power t o activate the ovum
if penetration into the vitellus is effected. We feel, however,
as shown in our study that the failure of activated or fertilized
ova to perform the first cleavage (as in the case of sperm
exposed to 65,00Or), to perform further cleavage at the 4
c ~ l stage
l
(as in the case of sperm treated with 6,500 r ) o r to
form a normal blastocyst (as in the case of sperm irradiated
at 800r) is the primary rather than a secondary effect of
irradiation of sperm.
With regard to the delayed syngamy in the ova fertilized
by sperm irradiated with the higher dose as stated by them,
if the syngamy means the contact of two pronuclei as shown
in their figure 6, plate 3, we have observed only 4 out of 144
ova in which pronuclei are not closely in contact with each
other at 8 hours after ovulation and this is not related to the
dose of treatment (table 2). They state further “the recovery
38 hours after ovulation of two ova, each with two polar bodies
and a central set of haploid chromosomes, suggests that completely normal fertilization did not occur in all ova at 100,000 r
(fig. 24, plate 6).” I n our material, fertilization and cleavage
did occur in 21 out of 59 ova at this dose level (table 1).
Furthermore in our experience a chromosome configuration as
shown in their figures 23 and 24, plate 6, is the typical chromosome configuration of an unfertilized ovum recovered 38
hours after ovulation. Penetration of spermatozoa into the
vitellus has never occurred. The two polar bodies mentioned
by them, may be two polar bodies cleaved from the first polar
body as shown in their figure 2, plate 3, because the second
polar body is morphologically different from the first polar
body (Chang, ’57).
As for the haploidy or triploidy of ova fertilized by irradiated sperm, we are not able to report confidently any such
occurrence in our materials. We feel that a description of a
IRRADIATION O F RABBIT SPE:RMATOZOA
221
triploid or haploid “chromosome complement’’ is not as
accurate as actual counting of chromosomes in a metaphase
plate. The occurrence of metaphase plates was scarce in the
early degenerated ova and they are diploid so far as we can
judge (figs. 13 and 17).
Irregularities in the blastomere nuclei of segmenting ova
such as rnultinucleate, small or large nuclei as well as anucleate
blastomeres (figs. 17,18a and 18b) were commonly observed in
the ova fertilized by irradiated sperm. It is, however, not
certain that these were due to the interference of nuclear
reformation as interpreted by Amoroso and Parkes (’47)
because the reformation of the nucleus is only one stage of
normal mitosis.
Since ionizing radiation affects the metabolism of nucleoprotein (cf. Patt ’53) and the synthesis of DNA (Errera, ’52),
the effect of radiation is more fundamental and is at a molecular rather than a morphological level. The morphological
abnormality of nucleus, spindle, chromosome, etc., caused by
irradiation of sperm is only a reflection of the damage done
to the nucleoprotein and the nucleic acids, which may not be
revealed by morphological observation. Thus, the disturbance
in the distribution of the chromosomes, the absence of a proper
division mechanism, or an atretic change in the ovum due to
delayed activation are only biological anticipations.
The gamma-irradiation of spermatozoa appears less harmful to embryonic development than X-irradiation because a low
percentage of fetal development (11%) and a relatively high
percentage of normal cleavage (55%) were obtained following
the irradiation of sperm at 800r while no implantation and
very low percentage of normal cleavage (2%) were observed
at this dose level (1,000 r ) by Amoroso and Parkes ( ’47). We
have had, however, no chance to determine the mortality of
these apparent normal young because they were examined
before delivery. Although our data show that low dosage
gamma irradiation appears not to interfere with embryonic
development no conclusions regarding the ultimate genetic
effects can be made.
222
M. C. CHANG, D. M. HUNT AND
E. B. ROMANOFF
After a review of literature on the effect of irradiation on
embryonic development, Rugh ( '53) stated that the newly
fertilized (amphibia) egg is more radiosensitive than either
gamete, as measured, not by survival, but by the effects on
embryonic development, and that radioresistance during the
first eight days of mammalian embryonic life is attributed to
the absence of differentiation rather than to the lack of
placental relation. In view of the extremely high radioresistance of rabbit spermatozoa to irradiation without damage to their fertilizing capacity, but with early degeneration
of fertilized ova, it seems that the fertilizing capacity of sperm,
at least the penetration into the vitellus, the formation of a
male pronucleus, and the activation of the ovum may involve
a biochemical system, which is not sensitive at all to irradiation, while the initiation of cleavage of the ovum and the
performance of normal mitosis may involve another biochemical system, which is much injured even in a resting
nucleus at the gamete stage. Although spermatozoa are not
at a stage of differentiation, if we assume that the cleavage
of ova is a phase of differentiation, then the latent damage
of irradiation on potential differentiation can be realized.
BUMMARY
1. Rabbit semen irradiated with gamma radiation from a
Co-60 source iw vitro at 90 r to 65,000 r was inseminated artificially to females. The ova were examined at pronuclear,
segmentation, blastocyst and late fetal stages.
2. The motility of spermatozoa was damaged only at very
high doses of irradiation, 32,200 to 65,000 r. Though incapacity
of sperm to reach the site of fertilization may occur at these
dose levels, the fertilizing capacity p e r se appears to be
undamaged.
3. The segmentation of fertilized ova was very much disturbed. The disturbance is in proportion to the dose level and
the higher the dose level the earlier the failure of segmentation. With irradiation of sperm at 90r, the proportion of
morphologically normal fetuses is not significantly lower than
IRRADIATION O F RABBIT SPERMATOZOA
223
after normal mating; following irradiation of sperm at 800 r
the formation of normal blastocysts was impaired but once a
normal blastocyst is formed implantation and development
are assured; following irradiation of sperm at 6,500 r, no
normal blastocyst is formed.
4. Delayed penetration of sperm, a high proportion of
polyspermic ova, haploidy and triploidy of ova, and delayed
syngamy were not observed following the irradiation of sperm
at 32,200 to 65,000r. Fragmentation. of female and male
pronuclei, an irregularity of nucleus formation at scgmentation, and one possible gynogcnetic ovum out of 32 ova were
observed.
LITERATURE CITED
ANOROSO,
E. C., A N D A. S. PARKES1947 Effects on embryonic development of
X-irradiation of rabbit spermatozoa in iritro. Proc. Roy. Soc. B, 134:
57-78.
c. R., AND
A. W. H. BRADEN 1953 An investigation of polyspermy in
the rat and rabbit. Aust. J. Biol. Sci., 6: 674492.
CHAW, M. C. 1950 Fertility and sterility as revealed in the study of fertilization and development of rabbit eggs. Fer. and Ster., 3: 205-222.
1952 Fertilizability of rabbit ova and the effects of temperature
in vitro on their subsequent fertilization and activation in vivo. J.
Exper. Zool., 121 : 351-381.
1954 Development of parthenogenetic rabbit blastocysts induced
by low temperature storage of unfertilized ova. J. Exp. Zool., 125:
127-150.
1955a The maturation of rabbit oocytes i n culture and their maturation, activation, fertilization and subsequent development i n the fallopian tubes. J. Exp. Zool., 128: 379-406.
1955b Fertilization and normal development of follicular oocytes in
the rabbit. Science, 121 : 867-869.
1957 Some aspects of mammalian fertilization. The beginnings of
embryonic development. AAAs Symposium. 109-134.
DARLINGTON,
C. D., AND L. F. LACOUR 1939 The Handling of Chromosomes.
George Allen and Unwin, London.
ERREBA,
M. 1952 Mecanismes de l’action des radiations sur le noyau cellulaire.
Ann. Soe. Roy. Sci. Med. et Nat. Bruxelles, 5: 65-176.
GREGORY,P. W. 1930 The early embryology of the rabbit. Contr. Embryol.
Carneg. Inst., 22 : 141-168.
JORDAN,
B. M., AND J. R. BAKER 1955 A simplie pyronine methyl green technique. Quart. J. Micro. Sci., 96: 177-179.
MACIRONE,
C., AND A. WALTON 1938 Fecundity of male rabbits as determined
122-134.
by “dummy matings.” J. Agr. Sci., ,988..
AUSTIN,
224
M.
c.
CHAW,
D. M. HUNT A N D
E. B. ROMANOFF
MURPHREE,R. L., F. J. WARWICX,L. F. CASIDA AND W. H. MCSHAN 1947
Influence of reproductive stage upon the fertility of gonadotrophintreated female rabbit. Endocrin., 41 : 308-312.
PATT,H. M. 1953 Radiation effects on mammalian systems. Ann. Rev. Physiol.,
16: 51-80.
RUGR, R. 1953 Vetebrate radiobiology: Embryology. AM. Rev. Nuc. Sci., 3:
271-302.
WALTON,A. 1930 The effect of temperature on the survival in vitro of rabbit
spermatozoa obtained from the vas deferens. J. Exper. Biol., 7:
201-219.
PLATES
PLATE 1
EXPLANATION OF FII3URES
All photographs taken under a phase contrast microscope
except figs. l b and 3b
l a A rabbit ovum fertilized by sperm irradiated a t 32,200 r, recovered about 8
hours after ovulation, photographed in saline showing the close cont.aot and
the normal appearance of two pronuclei a t this stage. X 150.
l b A group of ova fertilized by sperm irradiated at 65,000 r, recovered about 8
hours after ovulation, photographed after fixation showing the normal appearance of two pronuclei a t this stage. X 83.
2a Two ova fertilized by sperm irradiated at 65,00Or, recovered about 8 hours
after ovulation, photographed in saline showing one ovum with two well formed
pronuclei f a r apart and the close contact of pronuclei in the other ovum.
X 150.
2b The same ovum with pronuclei far apart as shown i n figure 2a. Photographed
after fixation. X 150.
a small fragment of female pronucleus fertilized by sperm
irradiated at 800 r, recovered about 8 hours after ovulation. X 150.
3a An ovum with
3b An ovum with a small fragment of female pronucleus fertilized by sperm
irradiated a t 6,500 r, recovered about 8 hours after ovulation, photographed
after fixation. X 255.
4
An ovum with several fragments of a male pronucleus fertilized by sperm
irradiated a t 6,500 r, recovered about 8 hours after ovulation. x 150.
5a An ovum with 3 pronuclei fertilized by sperm irradiated a t 38,200 r, recovered
about 8 hours ovulation. X 150.
5b The same ovum as shown in figure 5, photographed after fixation. X 150.
6
An ovum with only a female pronucleus fertilized by sperm irradiated a t
65,000 r, recovered 8 hours after ovulation, photographed after fixation.
X 150.
7
Two ova fertilized by sperm irradiated at 90 r, recovered about 38 hours after
ovulation, one ovuni cleaved to about 16 cells, the other about 8 cells. Photographed in saline. X 76.
8
Four ova fertilized by sperm irradiated a t 800r, recovered about 38 hours
after ovulation, two ova cleaved to about 8 cells, two ova cleaved to 4 t o 6
cells. X 76.
9 Four ova fertilized by sperm irradiated at 6,500 r, one ovum cleaved to about
8 cells, the others to about 4 cells. X 76.
226
IRR4DIATION OF RABBIT SPERMATOZOA
Xf.
C. CHAHG, D. M. HUXT
AND E. B. ItOYANOFF
2?i
PLATE I
PLATE 2
EXPLANATION OF FIGURES
All photographs taken under a phase contrast iiiicroscope except
figures 14 and 15
10
Five rabbit ova fertilized by Rperm irradiated a t G5,000r, recovered about
38 hrs. after ovulation, all ova a t 2 t o 3 cell stage, photographed in saline.
X 76.
11 One ovum with fused pronuclei b u t unclcaverl, fertilized by sperm irradiated
at 65,000 r, recovered about 38 hrs. after ovulation. Photographed in saline.
X 150.
12
One ovum fertilized by sperm irradiated a t 32,200 r, recovered about 38 hrs.
after ovulation and stained with acetic lacmoid, still a t the two-cell stage.
Nucleus with dark granules. X 150.
13
One ovum fertilized by sperm irradi:tted a t 65,000 r, recovrred about 38 hrs.
a f t e r ovulation and stained with arctic lacmoid showing chromosomes a t
metapliase b u t failnre t o cleave. X 150.
14
Kormal ( A ) and degenerated (R) blastoeysts recovered 6 days a f t r r the
insemimtioii of sperm irradiated a t 800 r. X 21.
15 Chromosome configuration of the germ disk of a hlastocyst recovered 6 days
after insemination with sperm irradiated :It 800 r. Feulgeii preparation.
Photographed with oil immersion objectivr. X 630.
16
Photograph of a degeiierated blnstocyst recovered 6 days a f t e r insemination
of sprrni irradiated a t 800 r. Stained with acetic lacmoid. X 160.
17
AII ovunl frrtilised by spcrm irradiated at 65,000 r, rcscovered about 38 hrs.
a f t e r ovulation. Stainrd with acctic lacmoid tilioming t h r niinclente cells
and the diploid configuration of c1ironiosomc.s. x 150.
18a 1Sb T’wo ova fertilized by sperm irradiated a t 65,000 r, recovered about
38 hrs. a f t e r ovulation. Photographed a f t e r fixatiou showing multinucleate
crlls and ovum fragmriits.
228
TRRADlATION O F RABRIT SPERMATOZ0.i
hl. C. CHANG, D. 3i. HUNT A N D E. 6.BOM-4NOFF
229
PLATE 2
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