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Sperm aging in utero and chromosomal anomalies in rabbit blastocysts
DEVELOPMENTAL
BIOLOGY
Sperm
28, 460-486 (1972)
Aging
in Utero
and Chromosomal
Rabbit PATRICIA Department
of Anatomy,
Accepted
in
Blastocysts
A. MARTIN University
Anomalies
AND EVELYN of Western Ontario, February,
L. SHAVER London,
Ontario,
Canada
25, 1972
Chromosomes were studied in 239 rabbit blastocysts recovered after aging of spermatozoa in utero. Mature females were inseminated artificially and induced to ovulate with an injection of HCG. The administration of HCG was delayed 4-21 hr after insemination, thus allowing sperm to age. Six-day blastocysts were recovered from females up to an estimated 30 hr sperm aging. There was a peak in the recovery rate at 18 hr and a decline thereafter. A significant difference was found between the incidences of chromosomal anomalies in the control and experimental series, where they were 1% and 9.7%, respectively. The abnormalities included mosaics, mixoploids, and XX/XY chimeras. The latter has been hitherto unreported in the rabbit. Dispermy with accompanying digyny has been implicated as the possible mode of origin of the chimeras. The sex ratio was not affected significantly by aging of spermatozoa. INTRODUCTION
Studies on a variety of vertebrates and invertebrates have demonstrated a gradual loss with time in the capability of the spermatozoa to effect fertilization and to support normal embryogenesis. Hammond and Asdell (1926) reported a marked reduction in fertility, as reflected in litter size, after rabbit spermatozoa had remained in the female tract for 22 hr before ovulation. After 30 hr, there were no litters. Tesh (1969) demonstrated that both preand post-implantation loss contribute to the decline in fertility in the rabbit. The losses appeared to occur at progressively earlier stages with increasing age of spermatozoa. Maurer et al. (1969) reported reduced cleavage rates after culturing embryos from rabbits inseminated 20 hours before ovulation. Cytological observations on the sperm penetration of rabbit eggs provided evidence that there is an increased incidence of dispermy when the interval between insemination and ovulation is 14 hours or longer (Harper, 1970a). The present investigation was conducted to study the consequences of aging rabbit spermatozoa on the chromosome constitution of resulting embryos. Such a 480 Copyright All rights
0 1972 by Academic Press, Inc. of reproduction in any form reserved.
study has not been undertaken for any animal and is particularly worthwhile considering that cleavage and fertilization errors are two major causes of chromosomal anomalies in embryos. MATERIALS
AND
METHODS
Forty mature females of New Zealand white and California strains weighing 2.9-4.6 kg were used in this study. All does were inseminated artificially and ovulation was induced by an intravenous injection of 60 IU HCG (‘A.P.L.’ Ayerst Laboratories). In the rabbit, ovulation occurs between 9.5 and 13 hr after an injection of HCG with a mean of 10 hr (Harper, 1963). Thirteen females served as controls and were given HCG at the time of insemination. In the experimental series, a delay in the administration of HCG until 4-21 hr post-insemination, resulted in an increased interval between insemination and ovulation thus allowing spermatozoa to age for 14-31 hr. The number of animals used at each time interval is given in Table 1. Semen was collected, by the use of an artificial vagina, from 9 bucks with a known high fertility. Immediately after collection, 0.1-0.2 ml of undiluted semen
MARTIN
TABLE
AND SHAVER
Sperm Age and Chromosomal
1
BLASTOCYSTS RECOVERED FROM RABBITS IN WHICH SPERMATOZOA WERE AGED in Utero
Aging period fFzafLs (hr) inseminated 10 (control) 14 16 18 20 22 25 28 29 30 31
Blastocysts recovered
Corpora lutea
Number
~
13 3 3 3 3 3 3 3 1 2 3
132 29 31 32 23 20 25 32 8 22 27 -
109 24 25 30 14 11 13 19 1 5 0 -
82.6 82.6 80.7 93.8 60.9 55.0 52.0 59.4 12.5 22.7 0
40
381
Total 251
containing 50 to 100 x lo6 spermatozoa with high motility was used to inseminate does in which sperm were aged for lo-28 hr. For spermatozoa aged 29-31 hr, 0.5 ml semen with a sperm count of approximately 250 x lo6 was used for each doe, The females were sacrificed 6-7 days after ovulation. Blastocysts were recovered by flushing the uterine horns with saline warmed to 37°C. The recovery rate was expressed as a percentage of blastocysts recovered to the number of corpora lutea counted in the ovaries. Chromosome spreads were made from each blastocyst by direct preparation, without culturing (Shaver and Carr, 1967). Slides were stained with Giemsa as outlined by Genest and Auger (1963). Metaphase plates were scored using bright field microscopy and photographs were taken for karyotypes. An analysis of the chromosomes in a minimum of 30 cells and the construction of 4 karyotypes from each blastocyst was the primary aim. When an abnormality was suspected, as many cells as possible were analyzed and additional karyotypes were made. Apart from numbering the autosomal pairs from 1 to 21, chromosomes were arranged ac-
481
Anomalies
cording to the karyotype and Benirschke (1967).
published
by Hsu
RESULTS
The percentage of ova released, as judged by the number of corpora lutea in the ovaries, and recovered as blastocysts at the various aging intervals is given in Table 1. The greatest recovery rate was observed when spermatozoa were aged 18 hr before the estimated time of ovulation. After this period, a decline occurred in the recovery rate with the most pronounced effect of sperm aging noticeable after 28 hr. No blastocysts were recovered from the 3 females inseminated with spermatozoa aged 31 hr. Every female ovulated resulting in a total of 381 corpora lutea with 251 blastocysts recovered 6-7 days later. The chromosomal complement was examined in 239 of the 251 or 95% of the blastocysts. Those blastocysts which could not be analyzed were extremely small and after processing were seen to have pycnotic nuclei. The sizes of analyzable blastocysts ranged from very large to small. A total of 14 blastocysts with detectable chromosomal defects emerged from this study. Table 2 presents the cytogenetic data for these heteroploid blastocysts. In the control series, 1 of 105 or 1% of the blastocysts analysed was chromosomally abnormal. This blastocyst, small in size, was a 2Nl4N mixoploid with a XXXX gonosomal complement in the tetraploid cell population. The remaining 13 heteroploid blastocysts, 5 mixoploids, 5 chromosome mosaics, and 3 sex chromosome chimeras, were observed in the experimental series giving a 9.7% incidence of chromosomal abnormalities. These anomalies were scattered among the various aging intervals with no increased frequency at any specific time. The 5 mixoploid blastocysts in the experimental series were very small. There was a marked contrast in size between
482
DEVELOPMENTAL BIOLOGY TABLE
VOLUME28. 1972
2
CYTOGENICFINDINGSIN HETEROPLOIDBLASTO~YSTS Chromosome Sperm aging periods (hr)
Diploid
<42 10 (control) 14
1 1
16 18
2 1 1 2
20 22 28
a) 4 b) 4 d 4 b) 4 b)
1
d
1 1 2
4 4 al b)
42
2
1
1 12
3
2 4 2 2
distribution Exact or near polyploid
and near diploid
43 2 12 2 1 10 24 1 3 2
1
1
2
4 5 13
4
count
44
45
46 47
19 26 15 13 47 15 33 20 I 14 1 72 3 4 36 3 45 9 3
these blastocysts and others recovered from the same female. In fact, mixoploids were by far the smallest blastocysts seen in the entire study. Diploid and tetraploid cell lines were found in each mixoploid. In addition, cells with an octoploid number of chromosomes were present in the mixoploid recovered from a female in which spermatozoa were aged for 18 hr. Another mixoploid, resulting from an ovum fertilized by spermatozoa aged 28 hr, had a cell line with 43 chromosomes. In this blastocyst, the missing chromosome was a submetacentric, possibly a homolog of pair 9. The sex chromosome complement was XXYY in the tetraploid cells of 3 ond XXXX in the remaining 2 mixoploid blastocysts. All the mosaic blastocysts, except one, were either large or medium-sized. The exception was the very small 44/45 mosaic recovered from a female in which spermatozoa were aged 20 hr. The extra chromosome in the 45 cell line of this blastocyst was found to be a large acrocentric. Two mosaics, a 43/44 and a 44/45, arose from ova fertilized by sperm aged 14 hr before ovulation and were found among blastocysts recovered from one rabbit. Karyotypes revealed the missing chromo-
No. cells karyotyped
SC3
8 8 15 9 16 17 6 3 3 6
8
14 12 9
xx XYIXO XY XXixY xx XY XY xx xx XY XXIXY XXIXY XY
9
9
XY
4N
EN
14
9 6 7 2
4
1
3
Anomaly
2Ni4N Mixoploid Mosaic Mosaic Chimera MOSt3iC
Mosaic 2N/4N Mixoploid 2Nf4N M&plaid 2Ni4N/8N Mixoploid Mosaic Chimera Chimera Hypodiploid mosaic/ mixoploid 43144188 2N/4N Mixoploid
TABLE
3
DISTRIBUTIONOF
CELLS IN THE CHIMERIC BLASTOCYSTS
Chimera
1 2 3
Sperm 53iw per,0.% (hr)
14 22 28
No. of cells with sex chromosomes identified
48 38 51
Sex chromosomes XY (55)
47.9 42.1 60
xx
P%)
52.1 57.9 40
some in the blastocyst with the 43 cell line to be possibly the Y chromosome and the additional chromosome in the blastocyst with the 45 cell line to be a small acrocentric. The remaining two blastocysts were 43144 chromosome mosaics arising from ova fertilized by sperm aged 16 and 18 hr. The missing chromosome was a large metacentric in both cases, the former lacking a homolog of pair 1 and the latter, a homolog of pair 3. Table 2 includes the sex chromosome complex of all the heteroploid blastocysts. The 3 chimeric blastocysts were recovered from females in which spermatozoa had aged 14, 22, and 28 hr. Two of these blastocysts were large and the other was medium-sized. Table 3 gives the distribution of cells with the XX and XY sex chromosome complement for each chimera. Karyotypes representing both cell popu-
MARTIN AND SHAVER
Sperm Age and Chromosomal
15 FIG. 1. Karyotype
7
of cell from XY population
16
17
of a chimeric
blastocyst.
7
8
15
14
FIG. 2. Karotype
483
Anomalies
of cell from XX population
lations from a chimeric blastocyst may be seen in Figs. 1 and 2. With the exception of the chimeras, the sex chromosome complement of all the blastocysts analyzed in this study is given in Table 4. In the control series, 51/105
17
16
in the same chimeric
blastocyst.
blastocysts had an XY and 54 an XX sex chromosome complex. This gave a sex ratio of 94 males to 100 females. The male sex chromosome complement was found in 80/134 blastocysts from the experimental series giving a sex ratio of 157 males/100
484
DET~ELOPMENTALBIOLOGY TABLE 4 CHROMOSOMAL SEX OF BLASTOCYSTP Series
Sperm aging period (hr)
No. of biastocysts analyzed cytogenetically
Sex complement E
P
Control
10
105/109
51
54
Experimental
14a 16 18 20 220 25 280 29 30
24/24 21/25 30130 12114 ll/ll 12/13 18/19 l/l 515
13 12 21 3 7 7 14 0 3
10 9 9 9 3 5 3 1 2
134/142 2391251
Grand total: ~The three chimeric this table.
blastocysts
0
are omitted
51 from
females. There was no significant difference between the control and experimental sex ratios (x2 = 3.20; 0.10 > P > 0.05). Also, when the sex ratio of the experimental group was compared with the hypothetical sex ratio of 100, the difference was not significant (x2 = 3.41; 0.10 > P > 0.05). DISCUSSION
This study is in accordance with previous investigators regarding a 30-hr life-span for rabbit sperm in utero (Hammond and Asdell, 1926; Tesh, 1969). However, we observed the first effect of sperm aging at 20 hr, which is 2 hr earlier than they reported. This discrepancy may be explained by variation in the time of ovulation between animals and among follicles within animals. The greatest recovery of blastocysts occurred at an estimated 18 hr sperm aging. This finding correlates with recent evidence of increased penetration of rabbit eggs when they enter the oviduct at the end of the ovulatory period and up to 19.5-20 hr after insemination (Harper, 1970b). It was suggested that this could be a reflection of a greater number of sperm
VOLUME 28, 1972
or of more fully capacitated sperm in the oviduct at the later time. The incidence of chromosomal anomalies in the experimental series was significantly different from the spontaneous incidence (x” = 6.7; P < 0.01). The results indicate that aging of sperm in the female tract has an adverse effect on the chromosome complement of resulting blastocysts. The changes which occur during the storage of sperm and which are responsible for functional inadequacy are as yet insufficiently known. The types and origin of the chromosomal anomalies observed in the blastocysts in this study, implicate both cleavage and fertilization errors as effects of aging sperm. A special feature of this investigation is the incidence of chimeras, which may occur from fertilization errors. Chimerism has not been previously observed in the rabbit. However, it is associated with intersexuality which has been reported in this species (Koch, 1963; Beatty, 1964; Shaver, 1967). The only reported chromosome study of intersex rabbits, revealed the presence of a normal XY chromosome complex (Shaver, 1967). However, this study was only in bone marrow and chimerism cannot be excluded. The mode of origin of chimerism is of considerable interest. Ford (1969) outlined double fertilization with aispermy and the participation of the ovum pronucleus and second polar body, as one of 2 main mechanisms giving rise to chimeras. The second mechanism which involves fusion of separate morulae would not be very feasible in rabbit embryos which have a thick mucin coat. Aging of rabbit sperm has resulted in dispermy (Harper, 197Oa) and digyny, the retention of the second polar body (Thibault, 1967). The simultaneous occurrence of digyny and dispermy with X- and Y-bearing sperm, after sperm aging, could result in double fertilization, giving rise to XX/ XY chimeric embryos with contributions from both fertilized polar body and ovum.
MARTIN AND SHAVER
Sperm Age and Chromosomal Anomalies
Aging of sperm resulted in a greater incidence of mixoploid blastocysts (5 of 134 or 3.7%) than in normal matings (1 of 75 or 1.3%) reported by Hofsaess and Meacham (1971). The tetraploid cells in the mixoploids probably originate from the failure of cytokinesis when karyokinesis occurs at the first or subsequent divisions of the zygote. This suppression of cytokinesis could explain the decreased blastocyst size observed in these mixoploids. While the significance of tetraploid cells in culture is doubtful, their occurrence in direct chromosome preparations, as employed in our investigation, reflects the true in viuo situation. The proportion of tetraploid cells in the mixoploids ranged from 22 to 44% as compared to 0.27% of total metaphases in all other blastocysts. The increased incidence of mixoploidy in our study can be correlated with the observation of Maurer et al. (1969) on the suppression of cleavage rates of embryos after sperm aging. Reports on 2N/4N mixoploids have been made from studies of human abortuses (Thiede and Salm, 1964) and live births (Kohn et al., 1967) and in mouse eggs after heat shock (Beatty and Fischberg, 1952). As in other studies of chromosomal aberrations in the rabbit, under different experimental conditions, the acrocentrics and large metacentrics were involved in aneuploidy (Shaver and Carr, 1967; Widmeyer, 1971; Hofsaess and Meacham, 1971). Distinction between small acrocentric autosomes and the Y chromosome is difficult in the rabbit. Therefore, the missing and additional small acrocentrics in two of the chromosome mosaics with an XY complement, cannot be established conclusively. The assessment of sex ratio in preimplantation embryos by the chmmosomal method gives a more reliable picture of the primary sex ratio than estimates on the morphological appearance of fetuses or live births. From this study, it appears that the sex ratio is not affected by the
485
length of time sperm are stored in utero. This is in accordance with Hammond and Asdell (1926) and Tesh (1969), who estimated the secondary sex ratio following aging of spermatozoa. The authors are grateful to Miss Isobel Morrison for technical assistance. This work was supported by grants from the Medical Research Council of Canada. REFERENCES BEATTY, R. A. (1964). Chromosome deviations and sex in vertebrates. In “Intersexuality in Vertebrates Including Man” (C. N. Armstrong and A. J. Marshall, eds.), pp. 1066115. Academic Press, New York. BEAM, R. A., and FISCHBERG, M. (1952). Heteroploidy in mammals. III. Induction of tetraploidy in preimplantation mouse eggs. J. &net. 50, 471-479. FORD, C. E. (1969). Mosaics and chimaeras. Bit. Med. Bull. 25, 1044109. GENEST, P., and AUGER, C. (1963). Observations on the technique for the study of human chromosomes by the culture of leucocytes from peripheral blood.
Can. Med. Ass. J. 88, 302-307. HAMMOND, J., and ASDELL, S. A. (1926). The vitality of the spermatozoa in the male and female reproductive tracts. &it. J. Exp. Biol. 4, 155-185. HARPER, M. J. K. (1963). Ovulation in the rabbit: The time of follicular rupture and expulsion of the eggs, in relation to injection of luteinizing hormone. J. Endocrinol. 26,307-316. HARPER, M. J. K. (1970a). Cytological observations on sperm penetration of rabbit eggs. J. Exp. Zool. 174, 141-156. HARPER, M. J. K. (1970b). Factors influencing sperm penetration of rabbit eggs in uiuo. J. Exp. Zool. 173, 47-62. HOFSAESS,F. R., and MEACHAM, T. N. (1971). Chromosome abnormalities of early rabbit embryos. J. Exp. Zool. 177,9-12. Hsu, T. C., and BENIRSCHKE, K. (1967). “An Atlas of Mammalian Chromosomes,” Vol. 1, p. 8. Springer-Verlag, Berlin and New York. KOCH, W. (1963). Intersexuality in mammals. In “Intersexuality” (C. Oversizer, ed.), pp. 35-47. Academic Press, New York. KOHN, G., MAYALL, B. H., MILLER, M. E., and MELLMAN, W. J. (1967). Tetraploid-diploid mosaicism in a surviving infant. Pediat. Res. 1,461-469. MAURER, R. R., WHITENER, R. H., and FOOTE, R. H. (1969). Relationship of in uiuo gamete aging and exogenous hormones to early embryo development in rabbits. PFOC.Sot. Exp. Biol. Med. 131,
882-885.
486
DEVELOPMENTAL BIOLOGY
SHAVER, E. L. (1967). Two cases of intersex in rabbits. Anat. Rec. 159, 127-130. SHAVER, E. L., and CARR, D. H. (1967). Chromosome abnormalities in rabbit blastocysts following delayed fertilization. J. Reprod. Fert. 14, 415-420. TESH, J. M. (1969). Effects of the ageing of rabbit spermatozoa in tltero on fertilisation and prenatal development. J. Reprod. Fed. 20, 299-306. THIBAULT, C. (1967). Analyse comparbe de la f&ondation et de ses anomalies chez la brebis, la vache
VOLUME 28, 1972
et la lapine. Ann. Biol. Anim. Biochim. Biophys. 7,5-23. THIEDE, H. A., and SALM, S. B. (1964). Chromosome studies of human spontaneous abortions. Amer. J. obstet. Gynecol. 90, 205-215. WIDMEYER, M. A. (1971) “Estrogen, Progesterone and Chromosome Abnormalities in Rabbit Biastocysts,” pp. 38. M.Sc. thesis, University of Western Ontario, London.
BIOLOGY
Sperm
28, 460-486 (1972)
Aging
in Utero
and Chromosomal
Rabbit PATRICIA Department
of Anatomy,
Accepted
in
Blastocysts
A. MARTIN University
Anomalies
AND EVELYN of Western Ontario, February,
L. SHAVER London,
Ontario,
Canada
25, 1972
Chromosomes were studied in 239 rabbit blastocysts recovered after aging of spermatozoa in utero. Mature females were inseminated artificially and induced to ovulate with an injection of HCG. The administration of HCG was delayed 4-21 hr after insemination, thus allowing sperm to age. Six-day blastocysts were recovered from females up to an estimated 30 hr sperm aging. There was a peak in the recovery rate at 18 hr and a decline thereafter. A significant difference was found between the incidences of chromosomal anomalies in the control and experimental series, where they were 1% and 9.7%, respectively. The abnormalities included mosaics, mixoploids, and XX/XY chimeras. The latter has been hitherto unreported in the rabbit. Dispermy with accompanying digyny has been implicated as the possible mode of origin of the chimeras. The sex ratio was not affected significantly by aging of spermatozoa. INTRODUCTION
Studies on a variety of vertebrates and invertebrates have demonstrated a gradual loss with time in the capability of the spermatozoa to effect fertilization and to support normal embryogenesis. Hammond and Asdell (1926) reported a marked reduction in fertility, as reflected in litter size, after rabbit spermatozoa had remained in the female tract for 22 hr before ovulation. After 30 hr, there were no litters. Tesh (1969) demonstrated that both preand post-implantation loss contribute to the decline in fertility in the rabbit. The losses appeared to occur at progressively earlier stages with increasing age of spermatozoa. Maurer et al. (1969) reported reduced cleavage rates after culturing embryos from rabbits inseminated 20 hours before ovulation. Cytological observations on the sperm penetration of rabbit eggs provided evidence that there is an increased incidence of dispermy when the interval between insemination and ovulation is 14 hours or longer (Harper, 1970a). The present investigation was conducted to study the consequences of aging rabbit spermatozoa on the chromosome constitution of resulting embryos. Such a 480 Copyright All rights
0 1972 by Academic Press, Inc. of reproduction in any form reserved.
study has not been undertaken for any animal and is particularly worthwhile considering that cleavage and fertilization errors are two major causes of chromosomal anomalies in embryos. MATERIALS
AND
METHODS
Forty mature females of New Zealand white and California strains weighing 2.9-4.6 kg were used in this study. All does were inseminated artificially and ovulation was induced by an intravenous injection of 60 IU HCG (‘A.P.L.’ Ayerst Laboratories). In the rabbit, ovulation occurs between 9.5 and 13 hr after an injection of HCG with a mean of 10 hr (Harper, 1963). Thirteen females served as controls and were given HCG at the time of insemination. In the experimental series, a delay in the administration of HCG until 4-21 hr post-insemination, resulted in an increased interval between insemination and ovulation thus allowing spermatozoa to age for 14-31 hr. The number of animals used at each time interval is given in Table 1. Semen was collected, by the use of an artificial vagina, from 9 bucks with a known high fertility. Immediately after collection, 0.1-0.2 ml of undiluted semen
MARTIN
TABLE
AND SHAVER
Sperm Age and Chromosomal
1
BLASTOCYSTS RECOVERED FROM RABBITS IN WHICH SPERMATOZOA WERE AGED in Utero
Aging period fFzafLs (hr) inseminated 10 (control) 14 16 18 20 22 25 28 29 30 31
Blastocysts recovered
Corpora lutea
Number
~
13 3 3 3 3 3 3 3 1 2 3
132 29 31 32 23 20 25 32 8 22 27 -
109 24 25 30 14 11 13 19 1 5 0 -
82.6 82.6 80.7 93.8 60.9 55.0 52.0 59.4 12.5 22.7 0
40
381
Total 251
containing 50 to 100 x lo6 spermatozoa with high motility was used to inseminate does in which sperm were aged for lo-28 hr. For spermatozoa aged 29-31 hr, 0.5 ml semen with a sperm count of approximately 250 x lo6 was used for each doe, The females were sacrificed 6-7 days after ovulation. Blastocysts were recovered by flushing the uterine horns with saline warmed to 37°C. The recovery rate was expressed as a percentage of blastocysts recovered to the number of corpora lutea counted in the ovaries. Chromosome spreads were made from each blastocyst by direct preparation, without culturing (Shaver and Carr, 1967). Slides were stained with Giemsa as outlined by Genest and Auger (1963). Metaphase plates were scored using bright field microscopy and photographs were taken for karyotypes. An analysis of the chromosomes in a minimum of 30 cells and the construction of 4 karyotypes from each blastocyst was the primary aim. When an abnormality was suspected, as many cells as possible were analyzed and additional karyotypes were made. Apart from numbering the autosomal pairs from 1 to 21, chromosomes were arranged ac-
481
Anomalies
cording to the karyotype and Benirschke (1967).
published
by Hsu
RESULTS
The percentage of ova released, as judged by the number of corpora lutea in the ovaries, and recovered as blastocysts at the various aging intervals is given in Table 1. The greatest recovery rate was observed when spermatozoa were aged 18 hr before the estimated time of ovulation. After this period, a decline occurred in the recovery rate with the most pronounced effect of sperm aging noticeable after 28 hr. No blastocysts were recovered from the 3 females inseminated with spermatozoa aged 31 hr. Every female ovulated resulting in a total of 381 corpora lutea with 251 blastocysts recovered 6-7 days later. The chromosomal complement was examined in 239 of the 251 or 95% of the blastocysts. Those blastocysts which could not be analyzed were extremely small and after processing were seen to have pycnotic nuclei. The sizes of analyzable blastocysts ranged from very large to small. A total of 14 blastocysts with detectable chromosomal defects emerged from this study. Table 2 presents the cytogenetic data for these heteroploid blastocysts. In the control series, 1 of 105 or 1% of the blastocysts analysed was chromosomally abnormal. This blastocyst, small in size, was a 2Nl4N mixoploid with a XXXX gonosomal complement in the tetraploid cell population. The remaining 13 heteroploid blastocysts, 5 mixoploids, 5 chromosome mosaics, and 3 sex chromosome chimeras, were observed in the experimental series giving a 9.7% incidence of chromosomal abnormalities. These anomalies were scattered among the various aging intervals with no increased frequency at any specific time. The 5 mixoploid blastocysts in the experimental series were very small. There was a marked contrast in size between
482
DEVELOPMENTAL BIOLOGY TABLE
VOLUME28. 1972
2
CYTOGENICFINDINGSIN HETEROPLOIDBLASTO~YSTS Chromosome Sperm aging periods (hr)
Diploid
<42 10 (control) 14
1 1
16 18
2 1 1 2
20 22 28
a) 4 b) 4 d 4 b) 4 b)
1
d
1 1 2
4 4 al b)
42
2
1
1 12
3
2 4 2 2
distribution Exact or near polyploid
and near diploid
43 2 12 2 1 10 24 1 3 2
1
1
2
4 5 13
4
count
44
45
46 47
19 26 15 13 47 15 33 20 I 14 1 72 3 4 36 3 45 9 3
these blastocysts and others recovered from the same female. In fact, mixoploids were by far the smallest blastocysts seen in the entire study. Diploid and tetraploid cell lines were found in each mixoploid. In addition, cells with an octoploid number of chromosomes were present in the mixoploid recovered from a female in which spermatozoa were aged for 18 hr. Another mixoploid, resulting from an ovum fertilized by spermatozoa aged 28 hr, had a cell line with 43 chromosomes. In this blastocyst, the missing chromosome was a submetacentric, possibly a homolog of pair 9. The sex chromosome complement was XXYY in the tetraploid cells of 3 ond XXXX in the remaining 2 mixoploid blastocysts. All the mosaic blastocysts, except one, were either large or medium-sized. The exception was the very small 44/45 mosaic recovered from a female in which spermatozoa were aged 20 hr. The extra chromosome in the 45 cell line of this blastocyst was found to be a large acrocentric. Two mosaics, a 43/44 and a 44/45, arose from ova fertilized by sperm aged 14 hr before ovulation and were found among blastocysts recovered from one rabbit. Karyotypes revealed the missing chromo-
No. cells karyotyped
SC3
8 8 15 9 16 17 6 3 3 6
8
14 12 9
xx XYIXO XY XXixY xx XY XY xx xx XY XXIXY XXIXY XY
9
9
XY
4N
EN
14
9 6 7 2
4
1
3
Anomaly
2Ni4N Mixoploid Mosaic Mosaic Chimera MOSt3iC
Mosaic 2N/4N Mixoploid 2Nf4N M&plaid 2Ni4N/8N Mixoploid Mosaic Chimera Chimera Hypodiploid mosaic/ mixoploid 43144188 2N/4N Mixoploid
TABLE
3
DISTRIBUTIONOF
CELLS IN THE CHIMERIC BLASTOCYSTS
Chimera
1 2 3
Sperm 53iw per,0.% (hr)
14 22 28
No. of cells with sex chromosomes identified
48 38 51
Sex chromosomes XY (55)
47.9 42.1 60
xx
P%)
52.1 57.9 40
some in the blastocyst with the 43 cell line to be possibly the Y chromosome and the additional chromosome in the blastocyst with the 45 cell line to be a small acrocentric. The remaining two blastocysts were 43144 chromosome mosaics arising from ova fertilized by sperm aged 16 and 18 hr. The missing chromosome was a large metacentric in both cases, the former lacking a homolog of pair 1 and the latter, a homolog of pair 3. Table 2 includes the sex chromosome complex of all the heteroploid blastocysts. The 3 chimeric blastocysts were recovered from females in which spermatozoa had aged 14, 22, and 28 hr. Two of these blastocysts were large and the other was medium-sized. Table 3 gives the distribution of cells with the XX and XY sex chromosome complement for each chimera. Karyotypes representing both cell popu-
MARTIN AND SHAVER
Sperm Age and Chromosomal
15 FIG. 1. Karyotype
7
of cell from XY population
16
17
of a chimeric
blastocyst.
7
8
15
14
FIG. 2. Karotype
483
Anomalies
of cell from XX population
lations from a chimeric blastocyst may be seen in Figs. 1 and 2. With the exception of the chimeras, the sex chromosome complement of all the blastocysts analyzed in this study is given in Table 4. In the control series, 51/105
17
16
in the same chimeric
blastocyst.
blastocysts had an XY and 54 an XX sex chromosome complex. This gave a sex ratio of 94 males to 100 females. The male sex chromosome complement was found in 80/134 blastocysts from the experimental series giving a sex ratio of 157 males/100
484
DET~ELOPMENTALBIOLOGY TABLE 4 CHROMOSOMAL SEX OF BLASTOCYSTP Series
Sperm aging period (hr)
No. of biastocysts analyzed cytogenetically
Sex complement E
P
Control
10
105/109
51
54
Experimental
14a 16 18 20 220 25 280 29 30
24/24 21/25 30130 12114 ll/ll 12/13 18/19 l/l 515
13 12 21 3 7 7 14 0 3
10 9 9 9 3 5 3 1 2
134/142 2391251
Grand total: ~The three chimeric this table.
blastocysts
0
are omitted
51 from
females. There was no significant difference between the control and experimental sex ratios (x2 = 3.20; 0.10 > P > 0.05). Also, when the sex ratio of the experimental group was compared with the hypothetical sex ratio of 100, the difference was not significant (x2 = 3.41; 0.10 > P > 0.05). DISCUSSION
This study is in accordance with previous investigators regarding a 30-hr life-span for rabbit sperm in utero (Hammond and Asdell, 1926; Tesh, 1969). However, we observed the first effect of sperm aging at 20 hr, which is 2 hr earlier than they reported. This discrepancy may be explained by variation in the time of ovulation between animals and among follicles within animals. The greatest recovery of blastocysts occurred at an estimated 18 hr sperm aging. This finding correlates with recent evidence of increased penetration of rabbit eggs when they enter the oviduct at the end of the ovulatory period and up to 19.5-20 hr after insemination (Harper, 1970b). It was suggested that this could be a reflection of a greater number of sperm
VOLUME 28, 1972
or of more fully capacitated sperm in the oviduct at the later time. The incidence of chromosomal anomalies in the experimental series was significantly different from the spontaneous incidence (x” = 6.7; P < 0.01). The results indicate that aging of sperm in the female tract has an adverse effect on the chromosome complement of resulting blastocysts. The changes which occur during the storage of sperm and which are responsible for functional inadequacy are as yet insufficiently known. The types and origin of the chromosomal anomalies observed in the blastocysts in this study, implicate both cleavage and fertilization errors as effects of aging sperm. A special feature of this investigation is the incidence of chimeras, which may occur from fertilization errors. Chimerism has not been previously observed in the rabbit. However, it is associated with intersexuality which has been reported in this species (Koch, 1963; Beatty, 1964; Shaver, 1967). The only reported chromosome study of intersex rabbits, revealed the presence of a normal XY chromosome complex (Shaver, 1967). However, this study was only in bone marrow and chimerism cannot be excluded. The mode of origin of chimerism is of considerable interest. Ford (1969) outlined double fertilization with aispermy and the participation of the ovum pronucleus and second polar body, as one of 2 main mechanisms giving rise to chimeras. The second mechanism which involves fusion of separate morulae would not be very feasible in rabbit embryos which have a thick mucin coat. Aging of rabbit sperm has resulted in dispermy (Harper, 197Oa) and digyny, the retention of the second polar body (Thibault, 1967). The simultaneous occurrence of digyny and dispermy with X- and Y-bearing sperm, after sperm aging, could result in double fertilization, giving rise to XX/ XY chimeric embryos with contributions from both fertilized polar body and ovum.
MARTIN AND SHAVER
Sperm Age and Chromosomal Anomalies
Aging of sperm resulted in a greater incidence of mixoploid blastocysts (5 of 134 or 3.7%) than in normal matings (1 of 75 or 1.3%) reported by Hofsaess and Meacham (1971). The tetraploid cells in the mixoploids probably originate from the failure of cytokinesis when karyokinesis occurs at the first or subsequent divisions of the zygote. This suppression of cytokinesis could explain the decreased blastocyst size observed in these mixoploids. While the significance of tetraploid cells in culture is doubtful, their occurrence in direct chromosome preparations, as employed in our investigation, reflects the true in viuo situation. The proportion of tetraploid cells in the mixoploids ranged from 22 to 44% as compared to 0.27% of total metaphases in all other blastocysts. The increased incidence of mixoploidy in our study can be correlated with the observation of Maurer et al. (1969) on the suppression of cleavage rates of embryos after sperm aging. Reports on 2N/4N mixoploids have been made from studies of human abortuses (Thiede and Salm, 1964) and live births (Kohn et al., 1967) and in mouse eggs after heat shock (Beatty and Fischberg, 1952). As in other studies of chromosomal aberrations in the rabbit, under different experimental conditions, the acrocentrics and large metacentrics were involved in aneuploidy (Shaver and Carr, 1967; Widmeyer, 1971; Hofsaess and Meacham, 1971). Distinction between small acrocentric autosomes and the Y chromosome is difficult in the rabbit. Therefore, the missing and additional small acrocentrics in two of the chromosome mosaics with an XY complement, cannot be established conclusively. The assessment of sex ratio in preimplantation embryos by the chmmosomal method gives a more reliable picture of the primary sex ratio than estimates on the morphological appearance of fetuses or live births. From this study, it appears that the sex ratio is not affected by the
485
length of time sperm are stored in utero. This is in accordance with Hammond and Asdell (1926) and Tesh (1969), who estimated the secondary sex ratio following aging of spermatozoa. The authors are grateful to Miss Isobel Morrison for technical assistance. This work was supported by grants from the Medical Research Council of Canada. REFERENCES BEATTY, R. A. (1964). Chromosome deviations and sex in vertebrates. In “Intersexuality in Vertebrates Including Man” (C. N. Armstrong and A. J. Marshall, eds.), pp. 1066115. Academic Press, New York. BEAM, R. A., and FISCHBERG, M. (1952). Heteroploidy in mammals. III. Induction of tetraploidy in preimplantation mouse eggs. J. &net. 50, 471-479. FORD, C. E. (1969). Mosaics and chimaeras. Bit. Med. Bull. 25, 1044109. GENEST, P., and AUGER, C. (1963). Observations on the technique for the study of human chromosomes by the culture of leucocytes from peripheral blood.
Can. Med. Ass. J. 88, 302-307. HAMMOND, J., and ASDELL, S. A. (1926). The vitality of the spermatozoa in the male and female reproductive tracts. &it. J. Exp. Biol. 4, 155-185. HARPER, M. J. K. (1963). Ovulation in the rabbit: The time of follicular rupture and expulsion of the eggs, in relation to injection of luteinizing hormone. J. Endocrinol. 26,307-316. HARPER, M. J. K. (1970a). Cytological observations on sperm penetration of rabbit eggs. J. Exp. Zool. 174, 141-156. HARPER, M. J. K. (1970b). Factors influencing sperm penetration of rabbit eggs in uiuo. J. Exp. Zool. 173, 47-62. HOFSAESS,F. R., and MEACHAM, T. N. (1971). Chromosome abnormalities of early rabbit embryos. J. Exp. Zool. 177,9-12. Hsu, T. C., and BENIRSCHKE, K. (1967). “An Atlas of Mammalian Chromosomes,” Vol. 1, p. 8. Springer-Verlag, Berlin and New York. KOCH, W. (1963). Intersexuality in mammals. In “Intersexuality” (C. Oversizer, ed.), pp. 35-47. Academic Press, New York. KOHN, G., MAYALL, B. H., MILLER, M. E., and MELLMAN, W. J. (1967). Tetraploid-diploid mosaicism in a surviving infant. Pediat. Res. 1,461-469. MAURER, R. R., WHITENER, R. H., and FOOTE, R. H. (1969). Relationship of in uiuo gamete aging and exogenous hormones to early embryo development in rabbits. PFOC.Sot. Exp. Biol. Med. 131,
882-885.
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DEVELOPMENTAL BIOLOGY
SHAVER, E. L. (1967). Two cases of intersex in rabbits. Anat. Rec. 159, 127-130. SHAVER, E. L., and CARR, D. H. (1967). Chromosome abnormalities in rabbit blastocysts following delayed fertilization. J. Reprod. Fert. 14, 415-420. TESH, J. M. (1969). Effects of the ageing of rabbit spermatozoa in tltero on fertilisation and prenatal development. J. Reprod. Fed. 20, 299-306. THIBAULT, C. (1967). Analyse comparbe de la f&ondation et de ses anomalies chez la brebis, la vache
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et la lapine. Ann. Biol. Anim. Biochim. Biophys. 7,5-23. THIEDE, H. A., and SALM, S. B. (1964). Chromosome studies of human spontaneous abortions. Amer. J. obstet. Gynecol. 90, 205-215. WIDMEYER, M. A. (1971) “Estrogen, Progesterone and Chromosome Abnormalities in Rabbit Biastocysts,” pp. 38. M.Sc. thesis, University of Western Ontario, London.