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Restriction fragment length polymorphism in the mitochondrial DNA of cloned cattle
Theriogenology 38:897-904.1992
RESTRICTION FRAGMENT LENGTH POLYMORPHISM IN THE MITOCHONDRIAL DNA OF CLONED CATTLE Yves Plant&” S.M. Schmutz’ and K.D.M. Lang’ ‘DNA Laboratory Saskatchewan Research Council Saskatoon, SK S7N 2X0 *Department of Animal and Poultry Science University of Saskatchewan, Saskatoon, SK S7N OWO Received for publication: March 20, 2992 Accepted: August 4, 1992 ABSTRACT The clonal origin of 4 Holstein bulls was determined by hybridization experiments with 2 different minisatellite probes, and all 4 animals showed identical genomic DNA fingerprints, hence confirming monozygosity. Extra-chromosomal differences were obsenred among these 4 Holstein bulls. Mitochondrial DNA restriction fragment length polymorphisms with restriction endonucleases hII and ml sites were found, and these polymorphisms can be explained by the loss of a single site for each of these 2 enzymes. Since mitochondtial DNA are maternally transmitted, all 4 bulls would produce genetically equivalent spermatozoa and offspring. The combination of embryo cloning and specific cytoplasmic markers would provide an ideal system for the study of maternal cytoplasmic effects on quantitative traits. Key words: embryo cloning, mtDNA, RFLP, cytoplasmic effect INTRODUCTION Embryo manipulation is increasingly used in modem cattle breeding programs. Multiple ovulation and embryo transfer (MOET) programs are now considered as practical methods for rapid genetic improvement of livestock (1,2). This particular methodology accelerates sire evaluation, thereby providing net
Acknowledgments We wish to thank M. Campos, T. Watts, and the Veterinary Infectious Disease Organization (VIDO) for sharing immunological data and providing the blood samples, and R. Mapletoft, P. Sabour, and A. Gibbins for valuable discussions. Funding was provided by the Saskatchewan Agriculture Development Fund.
Copyright 0 1992 Buttetworth-Heinemann
Theriogenology
898
economic gains to the cattle producers. Parallel to the MOET programs, embryo sexing (3-5) and embryo cloning (6) have also gained wide acceptance as methods to produce uniform stocks of desired animals and to allow more accurate evaluation of breeding values. Clones of cattle were first produced through embryo splitting (7). Twins are commonly produced by this procedure, and triplets and quadruplets are possible. More recently, nuclear transplantation has been advocated as the method of choice to produce large numbers of cloned calves (6). Velogenetics proposes to incorporate embryo manipulations and molecular biology technologies to decrease generation times in order to improve on genetic evaluation programs to be followed by the production of large numbers of cloned animals (9). Animals, within a clone, are assumed to have identical genotypes. With identical genetic background, these animals represent ideal material not only for uniform commercial cattle production (10) but also as experimental material in various fields such as drug and vaccine development, nutrition, and reproduction. In these particular areas of research, it is important to control genetic effects (maternal and paternal) in order to accurately assess the parameters, or the effects of environmental components, under study. The assumption of uniform maternal genetic background among the cloned animals may not always be true. The cloning procedure, by nuclear transplantation, usually involves removing and disrupting the inner cell mass from the early embryo to obtain isolated cells. These embryonic cells are then individually electrofused and transferred into enucleated mature oocytes (11). Hence there is transfer of the embryonic nucleus (and some embryonic cytoplasm) into a recipient cell containing a large amount of foreign cytoplasm. In most instances, oocytes are collected from slaughtered cows or from superovulated unrelated cows (11,12). In these particular cases, animals within a given clone will not share wmmon cytoplasm, and hence may differ because of various maternal cytoplasmic effects. To follow these effects, it is important to have unique cytoplasmic markers that will identify each animal within a given clone. Mitochondrial DNA (mtDNA) is a small circular genome which is maternally inherited (13). The bovine mtDNA has been sequenced (14), and restriction fragment length polymorphisms (RFLPs) have been reported (15-l 7). This small genome represents a unique molecular marker for cytoplasm. Here, we report on the analysis of mtDNA RFLPs found in a clone of 4 Holstein bulls obtained through nuclear transplantation. We also discuss a theoretical breeding approach to follow maternal cytoplasmic effects through the use of animal cloning and embryo transfer technologies. MATERIALS AND METHODS A clone of 4 and a second clone of 3 Holstein bulls were made available to us by the Veterinary Infectious Disease Organization (VIDO). Blood samples (7ml)
899
Theriogenology
were collected in EDTA vacutainer vials from the 2 clones of Holstein bulls. Buffy coats were recovered by centrifugation and washed several times in 10 mM TrisHCI (pH 7.5), 0.32 M sucrose, 5 mM MgCi,, 1% Triton X-l 00. The cells were then iysed in 4 M urea, 0.2 M NaCi, 0.1 M Tris-HCI (pH 8.0), 10 mM EDTA, 0.5% SDS, and proteinase K (10 pg). The iysates were incubated at 60°C for 16 hours. The iysates were then extracted several times with phenol and chloroform. The DNA was precipitated with 0.3 M sodium-acetate (pH 7.0) and 2 volumes of 95% ethanol, spooled onto a glass rod, washed in 70% ethanol, air dried, and resuspended in 10 mM Tris-HCI (pH8.0) and 1 mM EDTA. The DNA aiiquots (2 ug) were cleaved with &I, &I, &@I, &QHI, &!I, Mli, MElI, C&II, -RI, &gRV, Haelli. Hhai, HindIll, mfl, &l&i, &I, ml, &II, &I, &I, ul, l&l, ai, or ml. The DNA fragments were sizeseparated into 0.6% agarose gels at 2 V/cm, then they were vacuum-transferred onto nylon membranes (Hybond-N+, Amersham) using 0.4 N NaOH as the transfer buffer. These blots were prehybridized in 0.263 M N%HPO,, 7% SDS, 1% BSA, and 1 mM EDTA at 60°C for 16 hours. The Aiui, Haeili, mfl, and &+I blots were hybridized to minisatellite probe pV47-2 (18) and pSRC-15, a bovine minisatellite probe (Piante and Lang, unpublished data). Ail the blots were also hybridized to pAM1 (lS), a clone containing the entire mouse mtDNA (16,295 base pairs). The mtDNA sequence homology between the mouse and bovine genomes varies from 60 to 60% depending on the which blocks of the D-Loop and genes examined (20). The probes were radiolabeled by the random primer procedure (21). The blots were hybridized in the buffer described above. The membranes were washed in 2 X SSC, 0.5% SDS at room temperature twice for 15 minutes each, and in 0.5 X SSC, 0.5% SDS at 60°C twice for 30 minutes each. Blots were exposed to Kodak X-Omat AR films at -70°C for 16 - 24 hours. RESULTS Two minisatellite probes were used to verify the nuclear or chromosomai cionai origin of the Holstein bulls within the 2 clones. The probability that 2 full-sibs will share the same nuclear DNA banding patterns for these 2 minisatellites is less then 0.025. As predicted, Figure la shows that individuals Al, A2, A3, and A4 have identical banding patterns or DNA fingerprints, thus confirming that these animals are monozygotic twins or are from the same clone. individuals Bl ,B2, and 83 also have identical DNA fingerprints and are therefore shown to be from the second clone of bulls. Having established that these 4 cloned animals had identical nuclear backgrounds, extrachromosomai (cytopiasmic) similarities were examined. The mtDNA RFLPs were only detected within the clone of 4 bulls using restriction enzymes Av+l and ml. The polymorphisms involved the loss of a single Avall restriction site from mtDNA Type 1 to Type 2 and the loss of a single Hha_lssom mtDNA type 1 to type 2 (Figure 1b). Suptizingiy, these 2 mutations
Theriogenology
900
(mtDNA composite haplotype 2-1) were not detected in another sample of 19 cattle representing several breeds. Since partial digests can be ruled out, judged by the intensity and the total size of the bovine mtDNA fragments: and our procedure could not detect heteroplasmicity (22) these 4 animals are truly characterized by cytoplasms coming from different oocyte donor females.
A’
A2
A3
A” B’ B’
B3
A’ A2 A3 A” 10 7.1
8
5.0
6 3.6 2.9
4
1.4 1.2
1
a)
2
2
1
W
Figure 1. a) DNA fingerprints observed between the 2 clones of 4 (Al, A2, A3, and A4) and 3 (El, 82, and 83) Holstein bulls as revealed by minisatellite probe pSRC-15, the first lane on the right is a portion of a 1Kb molecular size standard. b) The mtDNA RFLPs at the ml sites {Type 1 or 2) among the clone of 4 bulls, sizes of the mtDNA fragments are indicated on the tight side.
Theriogenology
DISCUSSION The cattle industry has been extremely interested in the production of cloned animals with the hope of obtaining identical phenotypes. One obvious economic advantage is that only a single individual need be progeny proven and yet semen or oocytes (or embryos) would be available from other members of the clone which could be located in different countries. As shown by our RFLP data on a clone of 4 Holstein bulls obtained through nuclear transplantation, not all cloned individuals are identical in extrachromosomal genotypes. As expected, when more than 1 cow is collected for recipient oocytes to be enucleated, there is the potential for the mitochondrial genes within the clone of animals to differ, as has occurred in the clone of 4 Holstein bulls in this study. It should be noted, however, that the genetic contribution to all offspring of these 4 Holstein bulls will be identical, since the early embryonic cytoplasm and mitochondrlal genes are maternally inherited. Furthermore, we predict that these mitochondrial differences will occur relatively rarely, since it would appear that most cattle do not differ at most mitochondrial genes as evidenced by the lack of RFLPs in the 19 control animals. Although these mitochondtial differences may be perceived as a negative characteristic of cloned cattle by the industry, it allows for a unique opportunity to study maternal cytoplasmic effects on quantitative traits. Since many of the mitochondrial genes are involved in the energy cycle, it is possible that phenotypic traits such as growth and milk yield may be affected (23) by the origin of the recipient oocyte used during the cloning procedure. Here we will present a breeding approach using embryo transfer and animal cloning to specifically follow maternal cytoplasmic effects. Assuming fixed sire effects on the cloned progeny, we can describe at least two important maternal components in the progeny of a nuclear transplantation clone: (1) cytoplasmic effects of the recipient enucleated oocyte, and (2) in utero effects of the recipient cow. The in utero effects could be controlled for by cloning females from a single embryo. Sexing of the embryo can be achieved by the polymerase chain reaction (PCR) procedure using specific oligonucleotide primers (4). The female embryo can then be disrupted and isolated blastomeres electrofused to mature oocytes (11) collected from the same cow used to flushed the previous embryos. These reimplanted embryos would provide a clone of females with identical nuclear and cytoplasmic genomes. The procedure would assure that in utero effects will be fixed among the progeny produced from this clone of females. To study cytoplasmic effects, an early embryo would be disrupted and the blastomeres isolated. These cells will be used as donor nuclei to be transferred into enucleated mature oocytes collected from unrelated cows characterized by different mitochondrial genomes (RFLPs). These cows could also be from different breeds to maximize differences in cytoplasmic effects. The hybrid embryos could then be transferred into the cloned recipient cows. In this particular step of this breeding program, observed differences between individuals of the
902
Theriogenology
clone at birth would be strictly due to cytoplasmic effects. A similar approach can be designed to study in utero effects by fixing mitochondrial and cytoplasmic effects and mltochondrlal genes. This particular breeding program, relying on embryo cloning and embryo transfer would allow for the precise estimation of the various maternal effect components. Cytoplasmic effects are not necessarily a product solely of their organelles or DNA contained therein. The mitochondrial genome codes for subunits of some enzymes for which the other subunits are encoded in the nucleus. Much effort has gone into describing theoretical effects of such interactions (24), but empirical studies are limited (23,25). The breeding program proposed above would also shed more light on specific interactions existing between cytoplasmic and nuclear environments in developing embryos.
REFERENCES 1.
McGuirk, B. The relevance of MOET programmes to developing countries. Thetiogenology a:29-40 (1969).
2.
Wray, N.R. and Simm, G. The use of embryo transfer to accelerate genetic improvement in beef cattle. Proceedings of the 4’” World Congress of Genetics Applied to Livestock Production )(\11:334-346 (1990).
3.
Matthaei, K.I., Herr C.M., Bradley, M.P. and Reed K.C. Analysis of the sex chromosome constitution of single bovine spermatozoa by polymerase chain reaction (PCR). Theriogenology a:285 abstr. (1990).
4.
Schroder, A., Roschlau, D., Giehm, D., Schwerin, M. and Thomsen P.D. Sex determination of bovine DNA using Y-specific primers in the polymerase chain reaction. Arch. Tierz. 3:293-299 (1990).
5.
Herr, C.M. and Reed, KC. Micromanipulation of bovine embryos for sex determination. Theriogenology 35:45-54 (1991).
6.
Marx, J.L. Cloning sheep and cattle embryos. Science =:483-464
7.
Fehilly, C.B. and Willadsen, S.M. Embryo manipulation in farm animals. Ox. Rev. Reprod. Biol. &379-413 (1986).
6.
Willadsen, S.M. Nuclear transplantation in sheep embryos. Nature =:6365 (1986).
(1988).
Tberiogenology
903
9.
Georges, M. and Massey, J.M. Velogenetics, or the synergistic use of marker assisted selection and germ-line manipulation. Theriogenology 35:151-159 (1991).
10.
Smith, C. Cloning and genetic improvement of beef cattle. Anim. Prod. 9:49-62 (1969).
11.
Bondioli, K.R., Westhusin, ME. and Looney CR. Production of identical bovine offspring by nuclear transfer. Theriogenology a:1 65-l 74 (1990).
12.
Pieterse, MC., Vos, P.L.A.M., Kruip, Th.A.M., Wurth, Y.A., van Beneden, Th.H., Willemse, A.H. and Taveme, M.A.M. Transvaginal ultrasound guided follicular aspiration of bovine oocytes. Theriogenology 35:19-24 (1991).
13.
Gyllensten, U., Wharton, D., Josefsson, A. and Wilson, A.C. Paternal inheritance of mitochondrial DNA in mice. Nature =:255-257 (1991).
14.
Anderson, S., de Bruijn, H.L., Coulson, AR., Eperon, l.c., Sanger, S. and Young, LG. Complete sequence of bovine mitochondrial DNA. Conserved features of the mammalian mitochondrial genome. J. Mol. Biol. =:663-717 (1962).
15.
Watanabe, T., Masangkay, J.S., Wakana, S., Saitou, N. and Tomita, T. Mitochondrial DNA polymorphism in native Phillipine cattle based on restriction endonuclease cleavage patterns. Biochem. Genet. 27:431-436 (1969).
16.
Bhat, P.P., Mishra, B.P. and Bhat, P.N. Polymorphism of mitochondrial DNA (mtDNA) in cattle and buffaloes. Biochem. Genet. a:31 1-316 (1990).
17.
Koehler, C.M., Lindberg, G.L., Brown, DR., Beitz, D.C., Freeman, A.E., Mayfield, J.E. and Myers, A.M. Replacement of bovine mitochondrfal DNA by a sequence variant within one generation. Geneticsm:247-255 (1991).
16.
Longmire, J.L., Kraemer, P.M., Brown, NC., Hardekopf, L.C. and Deaven, L.L. A new multi-locus DNA fingerprinting probe: pV47. Nucleic Acids Research a:1 656 abstr. (1990).
19.
Martens, P.A. and Clayton, D.A. Mechanism of mitochondrial DNA replication in mouse L-cells: localization and sequence of the light-strand origin of replication. J. Mol. Biol. =:327-351 (1979).
20.
Southern, S.O., Souther P.J. and Dizon D.E. Molecular characterization of a cloned dolphin mitochondrial genome. J. Mol. Evol. a:32-42 (1966).
904
Theriogenology
21.
Feinberg, A.P. and Vogelstein, B. A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal, Biochem. m:266-267 (1984).
22.
Hauswirth, W.W., Van De Walle, M.J., Laipis, P.J. and Olivio, P.D. Heterogeneous mitochondrial DNA D-loop sequences in bovine tissue. Cell 37:1001-1007 (1984).
23.
Heindleder, S., Hecht, W. and Wassmuth, R. Characterization of ovine mitochondrial DNA and possible association of polymorphisms with phenotypic traits. Anim. Genet. 22(Suppl. 1):68 abstr. (1991).
24.
Asmussen, M.A., Arnold, J. and Avise, J.C. Definition and properties of disequilibrium statistics for association between nuclear and cytoplasmic genotypes. Genetics m:755-768 (1988).
25.
Alcivar, A.A. and Pineda, M.H. Differences in type of mitochondrial DNA (mtDNA) between donors and recipients do not affect pregnancy rates after transfer of rat embryos. Theriogenology 3:185 abstr. (1990).
RESTRICTION FRAGMENT LENGTH POLYMORPHISM IN THE MITOCHONDRIAL DNA OF CLONED CATTLE Yves Plant&” S.M. Schmutz’ and K.D.M. Lang’ ‘DNA Laboratory Saskatchewan Research Council Saskatoon, SK S7N 2X0 *Department of Animal and Poultry Science University of Saskatchewan, Saskatoon, SK S7N OWO Received for publication: March 20, 2992 Accepted: August 4, 1992 ABSTRACT The clonal origin of 4 Holstein bulls was determined by hybridization experiments with 2 different minisatellite probes, and all 4 animals showed identical genomic DNA fingerprints, hence confirming monozygosity. Extra-chromosomal differences were obsenred among these 4 Holstein bulls. Mitochondrial DNA restriction fragment length polymorphisms with restriction endonucleases hII and ml sites were found, and these polymorphisms can be explained by the loss of a single site for each of these 2 enzymes. Since mitochondtial DNA are maternally transmitted, all 4 bulls would produce genetically equivalent spermatozoa and offspring. The combination of embryo cloning and specific cytoplasmic markers would provide an ideal system for the study of maternal cytoplasmic effects on quantitative traits. Key words: embryo cloning, mtDNA, RFLP, cytoplasmic effect INTRODUCTION Embryo manipulation is increasingly used in modem cattle breeding programs. Multiple ovulation and embryo transfer (MOET) programs are now considered as practical methods for rapid genetic improvement of livestock (1,2). This particular methodology accelerates sire evaluation, thereby providing net
Acknowledgments We wish to thank M. Campos, T. Watts, and the Veterinary Infectious Disease Organization (VIDO) for sharing immunological data and providing the blood samples, and R. Mapletoft, P. Sabour, and A. Gibbins for valuable discussions. Funding was provided by the Saskatchewan Agriculture Development Fund.
Copyright 0 1992 Buttetworth-Heinemann
Theriogenology
898
economic gains to the cattle producers. Parallel to the MOET programs, embryo sexing (3-5) and embryo cloning (6) have also gained wide acceptance as methods to produce uniform stocks of desired animals and to allow more accurate evaluation of breeding values. Clones of cattle were first produced through embryo splitting (7). Twins are commonly produced by this procedure, and triplets and quadruplets are possible. More recently, nuclear transplantation has been advocated as the method of choice to produce large numbers of cloned calves (6). Velogenetics proposes to incorporate embryo manipulations and molecular biology technologies to decrease generation times in order to improve on genetic evaluation programs to be followed by the production of large numbers of cloned animals (9). Animals, within a clone, are assumed to have identical genotypes. With identical genetic background, these animals represent ideal material not only for uniform commercial cattle production (10) but also as experimental material in various fields such as drug and vaccine development, nutrition, and reproduction. In these particular areas of research, it is important to control genetic effects (maternal and paternal) in order to accurately assess the parameters, or the effects of environmental components, under study. The assumption of uniform maternal genetic background among the cloned animals may not always be true. The cloning procedure, by nuclear transplantation, usually involves removing and disrupting the inner cell mass from the early embryo to obtain isolated cells. These embryonic cells are then individually electrofused and transferred into enucleated mature oocytes (11). Hence there is transfer of the embryonic nucleus (and some embryonic cytoplasm) into a recipient cell containing a large amount of foreign cytoplasm. In most instances, oocytes are collected from slaughtered cows or from superovulated unrelated cows (11,12). In these particular cases, animals within a given clone will not share wmmon cytoplasm, and hence may differ because of various maternal cytoplasmic effects. To follow these effects, it is important to have unique cytoplasmic markers that will identify each animal within a given clone. Mitochondrial DNA (mtDNA) is a small circular genome which is maternally inherited (13). The bovine mtDNA has been sequenced (14), and restriction fragment length polymorphisms (RFLPs) have been reported (15-l 7). This small genome represents a unique molecular marker for cytoplasm. Here, we report on the analysis of mtDNA RFLPs found in a clone of 4 Holstein bulls obtained through nuclear transplantation. We also discuss a theoretical breeding approach to follow maternal cytoplasmic effects through the use of animal cloning and embryo transfer technologies. MATERIALS AND METHODS A clone of 4 and a second clone of 3 Holstein bulls were made available to us by the Veterinary Infectious Disease Organization (VIDO). Blood samples (7ml)
899
Theriogenology
were collected in EDTA vacutainer vials from the 2 clones of Holstein bulls. Buffy coats were recovered by centrifugation and washed several times in 10 mM TrisHCI (pH 7.5), 0.32 M sucrose, 5 mM MgCi,, 1% Triton X-l 00. The cells were then iysed in 4 M urea, 0.2 M NaCi, 0.1 M Tris-HCI (pH 8.0), 10 mM EDTA, 0.5% SDS, and proteinase K (10 pg). The iysates were incubated at 60°C for 16 hours. The iysates were then extracted several times with phenol and chloroform. The DNA was precipitated with 0.3 M sodium-acetate (pH 7.0) and 2 volumes of 95% ethanol, spooled onto a glass rod, washed in 70% ethanol, air dried, and resuspended in 10 mM Tris-HCI (pH8.0) and 1 mM EDTA. The DNA aiiquots (2 ug) were cleaved with &I, &I, &@I, &QHI, &!I, Mli, MElI, C&II, -RI, &gRV, Haelli. Hhai, HindIll, mfl, &l&i, &I, ml, &II, &I, &I, ul, l&l, ai, or ml. The DNA fragments were sizeseparated into 0.6% agarose gels at 2 V/cm, then they were vacuum-transferred onto nylon membranes (Hybond-N+, Amersham) using 0.4 N NaOH as the transfer buffer. These blots were prehybridized in 0.263 M N%HPO,, 7% SDS, 1% BSA, and 1 mM EDTA at 60°C for 16 hours. The Aiui, Haeili, mfl, and &+I blots were hybridized to minisatellite probe pV47-2 (18) and pSRC-15, a bovine minisatellite probe (Piante and Lang, unpublished data). Ail the blots were also hybridized to pAM1 (lS), a clone containing the entire mouse mtDNA (16,295 base pairs). The mtDNA sequence homology between the mouse and bovine genomes varies from 60 to 60% depending on the which blocks of the D-Loop and genes examined (20). The probes were radiolabeled by the random primer procedure (21). The blots were hybridized in the buffer described above. The membranes were washed in 2 X SSC, 0.5% SDS at room temperature twice for 15 minutes each, and in 0.5 X SSC, 0.5% SDS at 60°C twice for 30 minutes each. Blots were exposed to Kodak X-Omat AR films at -70°C for 16 - 24 hours. RESULTS Two minisatellite probes were used to verify the nuclear or chromosomai cionai origin of the Holstein bulls within the 2 clones. The probability that 2 full-sibs will share the same nuclear DNA banding patterns for these 2 minisatellites is less then 0.025. As predicted, Figure la shows that individuals Al, A2, A3, and A4 have identical banding patterns or DNA fingerprints, thus confirming that these animals are monozygotic twins or are from the same clone. individuals Bl ,B2, and 83 also have identical DNA fingerprints and are therefore shown to be from the second clone of bulls. Having established that these 4 cloned animals had identical nuclear backgrounds, extrachromosomai (cytopiasmic) similarities were examined. The mtDNA RFLPs were only detected within the clone of 4 bulls using restriction enzymes Av+l and ml. The polymorphisms involved the loss of a single Avall restriction site from mtDNA Type 1 to Type 2 and the loss of a single Hha_lssom mtDNA type 1 to type 2 (Figure 1b). Suptizingiy, these 2 mutations
Theriogenology
900
(mtDNA composite haplotype 2-1) were not detected in another sample of 19 cattle representing several breeds. Since partial digests can be ruled out, judged by the intensity and the total size of the bovine mtDNA fragments: and our procedure could not detect heteroplasmicity (22) these 4 animals are truly characterized by cytoplasms coming from different oocyte donor females.
A’
A2
A3
A” B’ B’
B3
A’ A2 A3 A” 10 7.1
8
5.0
6 3.6 2.9
4
1.4 1.2
1
a)
2
2
1
W
Figure 1. a) DNA fingerprints observed between the 2 clones of 4 (Al, A2, A3, and A4) and 3 (El, 82, and 83) Holstein bulls as revealed by minisatellite probe pSRC-15, the first lane on the right is a portion of a 1Kb molecular size standard. b) The mtDNA RFLPs at the ml sites {Type 1 or 2) among the clone of 4 bulls, sizes of the mtDNA fragments are indicated on the tight side.
Theriogenology
DISCUSSION The cattle industry has been extremely interested in the production of cloned animals with the hope of obtaining identical phenotypes. One obvious economic advantage is that only a single individual need be progeny proven and yet semen or oocytes (or embryos) would be available from other members of the clone which could be located in different countries. As shown by our RFLP data on a clone of 4 Holstein bulls obtained through nuclear transplantation, not all cloned individuals are identical in extrachromosomal genotypes. As expected, when more than 1 cow is collected for recipient oocytes to be enucleated, there is the potential for the mitochondrial genes within the clone of animals to differ, as has occurred in the clone of 4 Holstein bulls in this study. It should be noted, however, that the genetic contribution to all offspring of these 4 Holstein bulls will be identical, since the early embryonic cytoplasm and mitochondrlal genes are maternally inherited. Furthermore, we predict that these mitochondrial differences will occur relatively rarely, since it would appear that most cattle do not differ at most mitochondrial genes as evidenced by the lack of RFLPs in the 19 control animals. Although these mitochondtial differences may be perceived as a negative characteristic of cloned cattle by the industry, it allows for a unique opportunity to study maternal cytoplasmic effects on quantitative traits. Since many of the mitochondrial genes are involved in the energy cycle, it is possible that phenotypic traits such as growth and milk yield may be affected (23) by the origin of the recipient oocyte used during the cloning procedure. Here we will present a breeding approach using embryo transfer and animal cloning to specifically follow maternal cytoplasmic effects. Assuming fixed sire effects on the cloned progeny, we can describe at least two important maternal components in the progeny of a nuclear transplantation clone: (1) cytoplasmic effects of the recipient enucleated oocyte, and (2) in utero effects of the recipient cow. The in utero effects could be controlled for by cloning females from a single embryo. Sexing of the embryo can be achieved by the polymerase chain reaction (PCR) procedure using specific oligonucleotide primers (4). The female embryo can then be disrupted and isolated blastomeres electrofused to mature oocytes (11) collected from the same cow used to flushed the previous embryos. These reimplanted embryos would provide a clone of females with identical nuclear and cytoplasmic genomes. The procedure would assure that in utero effects will be fixed among the progeny produced from this clone of females. To study cytoplasmic effects, an early embryo would be disrupted and the blastomeres isolated. These cells will be used as donor nuclei to be transferred into enucleated mature oocytes collected from unrelated cows characterized by different mitochondrial genomes (RFLPs). These cows could also be from different breeds to maximize differences in cytoplasmic effects. The hybrid embryos could then be transferred into the cloned recipient cows. In this particular step of this breeding program, observed differences between individuals of the
902
Theriogenology
clone at birth would be strictly due to cytoplasmic effects. A similar approach can be designed to study in utero effects by fixing mitochondrial and cytoplasmic effects and mltochondrlal genes. This particular breeding program, relying on embryo cloning and embryo transfer would allow for the precise estimation of the various maternal effect components. Cytoplasmic effects are not necessarily a product solely of their organelles or DNA contained therein. The mitochondrial genome codes for subunits of some enzymes for which the other subunits are encoded in the nucleus. Much effort has gone into describing theoretical effects of such interactions (24), but empirical studies are limited (23,25). The breeding program proposed above would also shed more light on specific interactions existing between cytoplasmic and nuclear environments in developing embryos.
REFERENCES 1.
McGuirk, B. The relevance of MOET programmes to developing countries. Thetiogenology a:29-40 (1969).
2.
Wray, N.R. and Simm, G. The use of embryo transfer to accelerate genetic improvement in beef cattle. Proceedings of the 4’” World Congress of Genetics Applied to Livestock Production )(\11:334-346 (1990).
3.
Matthaei, K.I., Herr C.M., Bradley, M.P. and Reed K.C. Analysis of the sex chromosome constitution of single bovine spermatozoa by polymerase chain reaction (PCR). Theriogenology a:285 abstr. (1990).
4.
Schroder, A., Roschlau, D., Giehm, D., Schwerin, M. and Thomsen P.D. Sex determination of bovine DNA using Y-specific primers in the polymerase chain reaction. Arch. Tierz. 3:293-299 (1990).
5.
Herr, C.M. and Reed, KC. Micromanipulation of bovine embryos for sex determination. Theriogenology 35:45-54 (1991).
6.
Marx, J.L. Cloning sheep and cattle embryos. Science =:483-464
7.
Fehilly, C.B. and Willadsen, S.M. Embryo manipulation in farm animals. Ox. Rev. Reprod. Biol. &379-413 (1986).
6.
Willadsen, S.M. Nuclear transplantation in sheep embryos. Nature =:6365 (1986).
(1988).
Tberiogenology
903
9.
Georges, M. and Massey, J.M. Velogenetics, or the synergistic use of marker assisted selection and germ-line manipulation. Theriogenology 35:151-159 (1991).
10.
Smith, C. Cloning and genetic improvement of beef cattle. Anim. Prod. 9:49-62 (1969).
11.
Bondioli, K.R., Westhusin, ME. and Looney CR. Production of identical bovine offspring by nuclear transfer. Theriogenology a:1 65-l 74 (1990).
12.
Pieterse, MC., Vos, P.L.A.M., Kruip, Th.A.M., Wurth, Y.A., van Beneden, Th.H., Willemse, A.H. and Taveme, M.A.M. Transvaginal ultrasound guided follicular aspiration of bovine oocytes. Theriogenology 35:19-24 (1991).
13.
Gyllensten, U., Wharton, D., Josefsson, A. and Wilson, A.C. Paternal inheritance of mitochondrial DNA in mice. Nature =:255-257 (1991).
14.
Anderson, S., de Bruijn, H.L., Coulson, AR., Eperon, l.c., Sanger, S. and Young, LG. Complete sequence of bovine mitochondrial DNA. Conserved features of the mammalian mitochondrial genome. J. Mol. Biol. =:663-717 (1962).
15.
Watanabe, T., Masangkay, J.S., Wakana, S., Saitou, N. and Tomita, T. Mitochondrial DNA polymorphism in native Phillipine cattle based on restriction endonuclease cleavage patterns. Biochem. Genet. 27:431-436 (1969).
16.
Bhat, P.P., Mishra, B.P. and Bhat, P.N. Polymorphism of mitochondrial DNA (mtDNA) in cattle and buffaloes. Biochem. Genet. a:31 1-316 (1990).
17.
Koehler, C.M., Lindberg, G.L., Brown, DR., Beitz, D.C., Freeman, A.E., Mayfield, J.E. and Myers, A.M. Replacement of bovine mitochondrfal DNA by a sequence variant within one generation. Geneticsm:247-255 (1991).
16.
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