279

Mutation Research, 73 (1980) 279--290 © Elsevier/North-Holland Biomedical Press

THE INDUCTION AND DETECTION OF DIPLOID SPERMATOZOA IN Gallus domesticus

NANCY WANG * 1, JOHN R. SHEPPARD l, TINGCHUNG WANG 2 and ROBERT N. SHOFFNER 3

Department of Genetics and Cell Biology 1, Department of Surgery 2, and Department of Animal Science 3, University of Minnesota, Minneapolis, M155455 (U.S.A.)

(Received 11 April 1980) (Revision received 25 June 1980) (Accepted 1 July 1980)

Summary

Colcemid at the dose level of 0.37 mg/kg/day was injected intraperitoneally to 3 sexually active chicken males for 3 consecutive days. 10--12 days after the first colcemid injection, 14--25% of the sperm population in the semen samples from the treated males was found to be diploid in DNA content by flow micro- fluorometric analysis, Cytogeneic and developmental analyses on early embryos indicate that, during the process of spermatogenesis, the male germ cells are most susceptible to colcemid treatment 10--12 days prior to the maturation of the spermatozoa which is equivalent to the primary through secondary sperma- tocyte stages in chicken males. By the application of an extremely unequal chromosomal translocation as a cytological marker of parentage, it is confirmed that the diploid sperm induced are capable of uniting with a normal haploid or diploid egg to produce a triploid or tetraploid zygote.

Triploidy has been reported to occur spontaneously in the chicken (Gallus domesticus) (Abdel-Hameed, 1972; Abdel-Hameed and Shoffner, 1971; Bloom, 1970, 1972; Comings and Okada, 1971; Mong et al., 1974; Ohno et al., 1963), fish (Cimino, 1972; Dingerkus, 1976) and amphibians (Maxzon, 1977). Tri- ploidy may result from polyspermy, polygyny (the involvement of more than one egg in fertilization) or from the union of a normal, haploid gamete with a diploid one produced through chromosomal non~lisjunction during gameto- genesis. Experimentally, by suppressing meiotic svindle fiber formation in

* Present address: Depar tment of Laboratory Medicine and Pathology, College of Health Sciences, D-252 Mayo Memoztal Building, Univers/ty of Minnesota, Minneapolis, MN 55455 (U.S.A.).

280

oogenesis with colcemid, we have previously obtained, in the chicken, triploid zygotes which were comparable developmentally to diploids in both embryonic and post-hatched stages (Wang, 1978; Wang and Shoffner, 1972, 1980). Fur- thermore, the induction of diploid spermatids has been demonstrated by Tates (1979) and Tates et al. (1979) in Northern vole, Microtus oeconomus, with the treatment of methyl methanesulfonate, p-fluoroalanine and X-irradiation, resp., at the pre-spermatid stages.

The objectives of this study are: (1) to induce diploid spermatozoa through suppression of spindle fiber formation during spermatogenesis; flow micro- fluorometry is used to detect the existence and frequency of diploid sperm in the semen sample, (2) to determine if the induced diploid sperm can unite with a normal haploid egg to produce a polyploid zygote, a chromosomal rearrange- ment induced by the alkylating agent triethylene melamine (Wang, 1978) is used as a cytological marker to identify parentage, (3) to determine the male germ cell stage which is most sensitive to colcemid, and (4) to determine if gametic competition exists between diploid and normal haploid sperm by com- paring the frequency of diploid sperm detected by flow microfluorometry with the frequency of triploid zygotes identified in the early embryos.

Materials and methods

Mutagenic treatment A chromosomal rearrangement induced by the alkylating agent triethylene

melamine (Wang, 1978) provided a cytogenetic marker to identify parentage. In the chicken, the male is homogametic with ZZ sex chromosomes while the female is heterogametic with ZW sex chromosomes. 2 males homozygous for the unequal translocation t(Zq+; lq--) between chromosome 1 and the Z sex chromosome (TT--Z'Z') and one heterozygous (TN--Z'Z') male were used for the induction of diploid sperm. Colcemid (500 ~g/ml) was injected intraperi- toneally at a dose rate of 0.37 mg/kg/day, which is the dose used in chickens to arrest cells in mitosis for cytogenetic studies (Shoffner et al., 1967), for 3 con- secutive days. Each male was ejaculated prior to the first injection and daily after the injections for 4 days; Beginning the 5th day after the initial injection of colcemid, semen was' collected daily from each treated male for evaluation of mutagenic effects of colcemid at the gametic and early embryonic stages.

Flow microfluorometry (FMF) Variability in the DNA content of sperm is expected to be a sensitive indi-

cator of chromosomal numerical changes in spermatogenic cells. FMF is a promising system for determining the existence and frequency of diploid sperm because it allows cell analysis at extremely high rates with statistical precision and sensitivity not obtainable by other methods. The staining procedure of Krishan {1975) and Fried et al. (1976) was followed. A portion of the daily semen collection from each male was washed twice with Ringer's solution before staining with the DNA-binding fluorescent dye propidium iodide (0.05 mg/ml in 0.1% sodium citrate). When individually stained sperm cells flow single file across the laser beam, the dye fluoresces at an intensity which is pro- portional to the DNA content in each cell. In this manner a DNA content dis-

281

tribution can be obtained for the sperm population in the semen sample. Semen from an untreated rooster was used as a concurrent control.

Mating and incubation From the 5th to 59th day after the first colcemid injection, semen samples

were collected daily from each of the 3 treated males and used to inseminate 4 normal chromosome Leghorn females. Eggs were collected from each female for 10 days; therefore, a total of 30--40 eggs were collected for each sperm sample for a particular male. At the end of 10 days, the females were reused for a new insemination. All eggs collected for each semen sample were incubated for 48 h before excision for cytogenetic and developmental analyses. For the background control, semen was collected from the males 2 weeks prior to col- cemid injections, white leghorns were inseminated, and a 500-egg sample was evaluated for each male.

Early embryonic evaluation The 48-h embryos were harvested for chromosomal analysis according to the

methods of Shoffner et al. (1967). To accumulate metaphases, colcemid (0.01 ml of the 0.5% solution) was injected into the air cell above the embryos and incubation was continued for an additional 30--45 rain. The embryo was excised, transferred to a 0.45% (w/v) solution of sodium citrate for 10--15 rain of hypotonic treatment, fixed in 50% acetic acid and stored at --20°C until squash preparations were made. All preparations were screened in the phase- contrast microscope for heteroploidy; permanent slides were made of all sus- pect, abnormal embryos. After CO2 freezing to remove the cover slip, slides were immediately dehydrated in 2 changes of absolute alcohol, air~lried and stained with carbol fuchsin for further analysis of chromosome composition. For each embryo harvested, 25--30 metaphases were analyzed.

The percentage of dl (embryos which died before 48 h), fertility, aneuploidy and abnormal euploidy were calculated for the daily semen sample for each treated male. 500 eggs collected from the matings before colcemid injection were used as control for each individual male.

Results and discussion

Evaluation at the gametic level Beginning on the 5th day after colcemid injection, spermatozoa in the daily

semen sample from non-injected control and 3 treated males were analyzed for DNA content by FMF. The sperm population of the untreated male generated a narrow, unimodal DNA distribution (Fig. 1) indicating that the spermatozoa were homogeneous in morphology and haploid in DNA content.

There was a corresponding unimodal DNA distribution of sperm from each of the treated males from day 5 to day 9 after the first colcemid injection. Starting on the 10th day, however, sperm from the treated birds exhibited a bimodal distribution in DNA content. In addition to the main DNA peak seen with the control sperm, a minor satellite peak appeared. The peak channel position for the satellite (channel 55) was approx. 2-fold that of the main peak (channel 25), indicative of the existence of a sperm population with a DNA

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content double that of the main peak (Fig. 1). The channel number of the satellite D NA peak was almost identical for all 3 colcemid-treated males for each of the 3 days. During this period the semen quality was thin and watery as compared to the usual viscous, white appearance. By the 13th day, semen qua- lity returned to its normal appearance corresponding with the return of the sperm DNA to a unimodal distribution.

The satellite population, as calculated mathematically by the method of Fried et al. (1976) , represents 14--25% of the total spermatozoa in each semen sample (Table 1). The stallite peak is not a machine or preparation artifact because each day the control was prepared simultaneously and analyzed with the same calibration. It seems unlikely that the bimodal distribution is due to individual male variation, since each treated male gave a unimodal distribution

T A B L E 1

P E R C E N T A G E O F N A N D 2 N S P E R M IN T H E I N D I V I D U A L S E M E N S A M P L E A N A L Y Z E D B Y T H E

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283

before day 10 and after day 12. Judging from the peak channel position of the satellite, which is double that of the main peak, and the nature of mutagen applied, it is very likely that the satellite DNA distribution is contributed by a diploid sperm population induced by the spindle-fiber inhibitor. However, in order to find the real nature of the satellite DNA peak, it is necessary to isolate the spermatozoa from the 2 peaks by either differential centrifugation or the application of a cell sorter and to analyze each fraction individually for DNA content and biological function.

Biological evaluation at the embryonic level Embryos were harvested after 48 h of incubation and the percentage of

infertility, di (embryos which died before 48 h), aneuploidy and abnormal euploidy was recorded for each semen sample. To determine the mutagenic effect of colemid on the differential male germ cell stages, the percentage of infertility, d,, and abnormal euploidy were plotted against the days after colce- mid treatment (Fig. 2).

There were extreme peaks of infertility and d, embryonic mortality for each of the 3 treated males 10--12 days following the first colcemid treatment. This observation at the zygotic level correlates with the flow microfluorometry analysis which shows a peak of diploid gametes 10--12 days following the first colcemid injection for each of the 3 treated males. The possible explanation for the high frequency of infertility is that inhibition of microtubule formation at M, or M2 may have interfered with the subsequent spermiogenesis or fertiliza- tion. Peak frequency of abnormal euploidy (haploids, triploids and tetraploids) was also around 11 days following injection (Fig. 2). These observations sug- gested that between 10--12 days before maturation, the male germ cells are most sensitive to colcemid treatment.

Totally, there were 17 haploids and 16 polyploids obtained. Using the un- equally translocated chromosome as a cytogenetic marker, it was possible to identify parentage of all the abnormal euploids obtained from progeny of the translocation homozygous males. Genome composition of the haploids was found to be all of male origin with a chromosome constitution of T'--Z'. A possible explanation is that these haploid cells originate from sperm nuclei sur- rounding the true embryos which died at an extremely early stage (Snyder et al., 1975). The chromosome compositions of the 16 polyploids are listed in Table 2. As shown, there were 5 polyploids of male or male and female origin with chromosome compositions of TTNN--Z'Z'ZZ, TTN--Z'Z'W {Fig. 3), TTN--Z'Z'ZZ and TTNN--Z'Z'WW (Fig. 4). The appearance of 2 translocated chromosomes, TT or Z'Z', confirms the male origin of the diploid gamete. It is unlikely that the polyploid zygotes were originated from dispermy, since it is hard to rationalize the induction of dispermy during fertilization with the colcemid treatment during spermatogenesis in the male parent. These. poly- ploids resulted from insemination on days 8, 11, 13, 16 and 20, resp., and represent 14.3%, 50%, 12.5%, 7.7% and 6.3% of the viable embryos recovered from the same insemination. The data suggest that a diploid sperm may be induced by colcemid and the induced diploid sperm can unite with either a haploid or diploid egg to produce a triploid or tetraploid zygote which is devel- opmentally compatible up to the 48-h stage.

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As also shown in Table 2, 4 triploids with a chromosome composi t ion of TNN were found in the progeny of translocation homozygous males (males Nos. 1 and 3). Appearance of only one translocated (T) chromosome indicates that the origin of the diploid gamete is from the female rather than the male parent. Judging from the sex chromosome composi t ion of the 4 triploids, the 2 ZZZ and 1 ZWW triploids are a result of chromosomal nondisjucntion at M2, and the ZZW triploid is a result of nondisjunction at M1 of oogenesis. Totally 8 polyploids, 7 triploids and 1 tetraploid, were recovered from the progeny in the translocation heterozygous male (male No. 20). The tetraploid, with a chromo- some composit ion of TTNN--Z 'Z 'ZZ, resulted from the union of a diploid sperm (TT--Z'Z') and a diploid egg (NN--ZZ) while the triploid, with a chro- mosome composi t ion of NNN--ZWW, resulted from the union of a diploid egg (NN--WW) with a haploid sperm (N~Z). The rest of the triploids have an auto- some composi t ion of either TNN or NNN and a sex-chromosome composi t ion of either ZZZ or ZZW; therefore, parentage of the diploid gametes is not iden- tifiable. No polyploid was found in the progeny of the controls.

In addition to abnormal euploidy, the formation of aneuploidy was also used

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Fig. 4. Metaphase spread of the tetraploid chicken embryo with a chromosome compos i t ion of TTNN-- Z'Z'WW.

as an evaluation criterion for mutagenicity. Percentage of aneuploidy was plotted against the days after colcemid treatment. As shown in Fig. 5, fre- quency of the aneuploidy is much higher in progeny of the translocation het- erozygous male either with or without colcemid treatment. Furthermore, there is no specific pattern in the distribution of aneuploids with respect to the time of colcemid injection. All observed aneuploids were tertiary trisomy of either chromosome 1 or the Z sex chromosome, both of which are involved in the translocation. The high incidence of nondisjunction in translocated chromo- somes of the heterozygous male, therefore, appears more as an intrinsic prop- erty of the unequal chromosomal rearrangement rather than a consequence of colcemid treatment. The positive association of aneuploidy or meiotic chromo- somal nondisjunction with tmnslocation heterozygosity has been previously reported in the murine system (De Boer, 1973; Gropp et al., 1974; White et al., 1974).

In this study we have demonstrated by flow microfluorometry that diploid spermatozoa could be induced through the suppression of meiotic spindle fiber formation by colcemid during spermatogenesis. Furthermore, it is evident by the application of an equal translocation as a cytological marker of parentage that the diploid sperm are capable of uniting with a haploid or diploid egg to produce a polyploid zygote. Both gametic and zygotic analyses indicate that the male germ cell stages which are most sensitive to colcemid inhibition were about 10--12 days prior to maturation of the spermatozoa, which is equivalent to the primary through secondary spermatocyte stages in chicken male

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(Takeda, 1969). These data suggest that the meiotic spindle-fiber formation in chicken males probably occurs in this period of spermatogenesis. However, due to the high frequency of infertility and dl embryonic mortality caused by colcemid during the peak effective period (see Fig. 1), only a small number of viable zygotes (Totally 11 embryos recovered during the period of day 10--12) were available for cytogenetic analysis. Consequently, no definite conclusion can be made about the gametic competition between diploid and normal hap-

289

loid sperm. In order to determine the selective difference between 2N and 1N sperm at the gametic, embryonic and hatching level, a large-scale study including FMF analysis, early embryonic cytogenetic analysis and hatching data analysis may be performed during the peak effective period of the drug. More- over, it is probably beneficial to apply colcemid by either a single high<lose injection or multiple injections of lower doses to avoid the toxicity effect of the drug.

Acknowledgements

We wish to thank Jack Otis, Ruth Shuman and Bill Raux for their skilled technical assistance, Kate Perkins and Diane Konzen for critical reading of the manuscript.

The effort of the Merck, Sharpe and Dohme Research Laboratories is grate- fully acknowledged.

Scientific Journal Series No. 11204 of the Minnesota Agricultural Experi- ment Station.

References

Abdel-Hameed, F. (1972) Hemoglobin concentration in normal diploid and tntersex triploid chickens: Genetic inactivation or canalization? Science, 178, 864.

Abdel-Hameed, F., and R.N. Shoffner (1971) Intersexes and sex determination in chickens, Science, 172, 962---964.

Bloom, S.E. (1970) Trtsomy-3-4 and triploldy (3A-ZZZW) in chicken embryos: Autosomal and sex chro- mosomal nondisjunction in meiosis, Science, 170, 457--458.

Bloom, S.E. (1972) Chromosome abnormalities in chicken (Gallus domesticus) embryos: Types, fre- quencies and phenotypic effects, Chromosoma, 37,309--326.

Borisy, G.G., and E.W. Taylor (1967) The mechalnsm of action of colchicine, Binding of colchicine-3H to cellular protein, J. Cell Biol., 34, 525--533.

Cimino, M.C. (1972) Meiosis in triploid all-female fish (Poeciliopsis poeciliidae), Science, 175, 1484-- 1486.

Comings, D.E., and R.A. Ikada (1971) Triple chromosome pairing in triploid chickens, Nature (London), 231,119--121.

De Boer, P. (1973) Fertile tertiary trisomy in the mouse (Mus musculus), Cytogenet. Cell Genet., 12, 435--442.

Dingerkus, G. (1976) Karyotypic analysis and evidence of tetraploidy in the North American Paddleflsh, Polyodon spathula, Science, 194,842--844.

Fried, J. (1975) Mathematical analysis of flow mierofluorometric data and its application to the termina- tion of cell cycle parameters, in: A.J. Valleron (Ed.), Mathematical Models in Ceil Kinetics, European Press, Ghent, Belgium, pp. 68--70.

Fried, J., A.G. Perez and B.D. Clarkson (1976) Flow cytofluorometric analysis of cell cycle distribution using propidinm iodide, J. Cell Biol., 71,172--181.

Gropp, A., D. Giers and U. Kolbus (1974) Trisomy in the fetal backcross progeny of male and female metacentric heterozygotes of the mouse, I. Cytogenet. Cell Genet., 13,511--535.

Krishan, A. (1975) Rapid flow cytpfluorometric analysis of mammalian cell cycle by propidium iodide staining, J. Cell Biol., 66, 188--193.

Maxson, L. (1977) Immunological resolution of a diploid---tetraploid species complex of the frog, Science, 197, 1012--1013.

Mong, S.J., M.D. Snyder, N.S. Feichhcimer and R.G. Japp (1974) The origin of triploidy in chicken (Gallus domesticus) embryos, Can. J. Genet. Cytol., 16, 317--322.

Ohno, S., W.Z. Kitreil, L.C. Christian, C. Stentus and G.A. Witt (1963) An adult triploid chicken (Gallus domesticus) with a left ovotestis, Cytogenetics, 2, 42--49.

Shoffner, R.N., A. Krishan, G.J. Halden, R.K. Bammi and J.S. Otis (1967) Avian chromosome methodol- ogy, Poultry Sci., 46, 333--334.

Snyder, M.D., N.S. Fechheimer and R.G. Jaap (1975) Incidence and origin of heteroploidy espe'cially haploidy in chick embryos from intraline and interline matings, Cytogenet. Cell Genet., 14, 63--75.

2 9 0

T a k e d a , A. ( 1 9 6 9 ) Labe l l i ng o f c o c k s p e r m a t o z o a w i t h r a d i o a c t i v e p h o s p h o r u s , J p n . J . Z o o t e c h . Sci. , 40 , 4 1 2 .

Ta tes , A .D . ( 1 9 7 9 ) M i c r o t u s oeconornus ( R o d e n t i a ) , a use fu l m a m m a l f o r s t u d y i n g the i n d u c t i o n o f sex- c h r o m o s o m e n o n d i s j u n c t i o n a n d d i p l o i d g a m e t e s in ma le g e r m cells, E n v i r o n . H e a l t h Pe r spec t . , 31 , 1 5 1 - - 1 5 9 .

Tares , A .D . , P .L . P e a r s o n , M. van d e r P loeg a n d H. de Voge l ( 1 9 7 9 ) The i n d u c t i o n o f s e x - c h r o m o s o m a l n o n d i s j u n c t i o n a n d d ip lo id s p e r m a t i d s f o l l o w i n g X - i r r a d i a t i o n or p r e - s p e r m a t i d s tages in the N o r t h e r n vole , M i c r o t u s oeconornus , M u t a t i o n Res . , 61 , 8 7 - - 1 0 1 .

Wang , N. ( 1 9 7 8 ) I n d u c t i o n o f c h r o m o s o m a l s t r u c t u r a l a n d n u m e r i c a l c h a n g e s in the c h i c k e n (Gallus dornes t icus ) , Ph .D. Thes is , Un ive r s i ty o f M i n n e s o t a .

W a n g , N. , a n d R .N . S h o f f n e r ( 1 9 7 2 ) I n d u c e d h e t e r p l o i d y in Gallus d o m e s t i c u s , Gene t i c s , 7 1 ( 2 ) , 66 . W a n g , N. , a n d R .N . S h o f f n e r ( 1 9 8 0 ) I n d u c t i o n o f h e t e r o p l o i d y in GaUus d o m e s t i c u s , M u t a t i o n Res . , 69 ,

2 6 3 - - 2 7 3 . Whi t e , B .J . , J . H . Tj io , L.C. van de W a t e r a n d C. Cranda l l ( 1 9 7 4 ) T r i s o m y 19 in the l a b o r a t o r y m o u s e , I.

F r e q u e n c y in d i f f e r e n t c rosses a t spec i f i c d e v e l o p m e n t a l s tages a n d r e l a t i o n s h i p o f t r i s o m y of c le f t pa l a t e , C y t o g e n e t . Cell G e n e t . , 13 , 2 1 7 - - 2 3 1 .