Micromanipulation with embryos of animals




Obtaining identical twins. The basic premise of the natural manifestations of multiple pregnancy in mammals is the simultaneous fertilization of at least two mature eggs by different sperm. Cattle is characterized by low twins   frequency (an average of 0,025). Among calves- twins are sometimes found identical twins. The likelihood of such genetically identical twins is only 0.01%. Low rates of twins   incidence and heritability do not allow to expect the high efficiency of selection. Therefore of great practical importance is the  genotypes copy methods of highly productive animals on the basis of early embryo separation by  microsurgery and micromanipulation techniques  into two or more blastomeres capable to develop  during the entire ontogeny, ie capable to express their totipotency. The possibility micromanipulation with individual embryos was proven in 1936 G Pincus. He introduced a single blastomeres of 2-cell rabbit embryo in  oviduct of  falsely pregnant rabbit. Embryos developed normally with differentiation of their cells to blastocyst stage (stages 2, 4 and 8 blastomeres, morula stage,  blastocyst stage). Then offspring of rabbits and mice was received from two-cell embryos (stage 2 blastomeres) with the destruction of a single blastomere by puncturing it with a needle. These studies were the basis for the further improvement of micromanipulation technique with embryos of animals, and in particular to obtain identical twins. The latter is of great importance to  intensification of livestock, since it facilitates the increase of output young animals from a single donor and allows to get genetically identical twins. SMWilladsen first reported in 1979 about getting of monozygotic twins in sheep by dividing 2-cell blastomere. He divided blastomeres into two separate cells, and impaired pellyutsida area (pellyutsida is formation  preventing spillage of blastomeres, as well as contact with other embryos, foreign cells, white blood cells, sperm cells and facilitates the passage of the embryo through oviduct  cloged with agar. Enclosing separated blastomeres in agar, which is practically insoluble in the female genital tract, allowed them to survive and develop in vivo. For the cultivation of embryos enclosed in agar sheep  was used as temporary recipients (sheep oviduct is the most suitable object for cows, horses and pigs embryo development up to blastocyst). Survival rate of "halves" of embryos at this stage after  transplantation to recipients was about 50%.

In sheep, two-cell blastomeres can be obtained in a very short period of time. So, after 60 hours, most embryos are on the 4-cell stage of development. Due to significant fluctuations of  intervals between injections of  pregnant mare serum (PMS) and the beginning of hunting, between hunting season and the beginning of ovulation, it is almost impossible during the operation of sheep to find embryos, which are on the 2-cell stage. Later experiments showed that genetically identical twins can also be obtained from the 4 - and 8 - cell blastomeres by splitting them into two groups. These "half" were equally viable as a normal sheep embryos. It is established that embryos derived from  8-cell blastomeres have no vitality. It is believed that the sharp decrease in the number of cells of the embryo is a major factor reducing their ability to develop into viable blastocysts.

 Technique of  enclosing in agar blastomeres of cattle embryos divided into parts to get  identical twins was successfully used by SMWilladsen et al. (1981) in obtaining  calves - identical twins. Investigations were carried out on 5-6-day-old embryos at morula stage, because in cows non-surgically method  more appropriate to get embryos. Morula were divided in "half" or "quarters", enclosed in agar and transferred to the oviduct of anestralnoy sheep for 1-2 days. Then they were removed and surgically transplanted to recipients on the 6th and 7th day of the sexual cycle. Engraftment of "halves" was high (75%), while this figure in "quarters" was significantly lower (41%). The authors concluded that the ability of splitting cow embryo to regulate their development in the case of  decreasing the number of cells is largely preserved even at morula stage. W.R. Allen and R.L. Pashen (1984) in experiments on horses proved the possibility of obtaining identical twins in the same manner using 2-8-cell blastomeres. G.Brem et al. (1983) have developed a technology for the production of  identical twins using a 6-day-old embryos (morulae) received by non-surgical way. Embryos fixed with pipettes. With the help of micro knife pellyutsida zone was cut, and then incision was opened by glass needle. Micro knife or glass thread was introduced through a hole to cut morula into two halfs. One half of the embryo left in pellyutsida’s own zone, and the other was placed in a transparent area of  unfertilized cattle oocytes. According to the authors, engraftment of separated embryos was 60%. From the all implanted embryos 50% of the pairs of twins were received, one out of three divided embryo gave a pair of twins. Subsequent experiments on the production of monozygotic twins have been focused on the use of late morulae and blastocysts, because at that stages of development protection from the pellyutsida zone is not insignificant. SMWilladsen and RAGodke (1984) carried out  separation of sheep embryos at the late morula stage, at the stage of early, late and hatching blastocyst into two equal halves.

In this case  part of the halves of embryos remained inside pellyutsida torn areas, while others were transplanted without it. Halves of embryos were transplanted to the same sheep from that they have been removed. 16 out of 18 sheep lambing, among them 7 heads gave birth to one lamb, 8 heads - to identical twins and one head gave birth to no monozygotic twins. Moreover, the value of  zone pellyutsida in the development of separated blastocysts is not revealed. Engraftment of embryos’ halves on the  stage of late morula and early blastocyst was almost two times lower (42%) than in the late stage or in the stage of hatched blastocysts (79%). Researchers have not received any single egg twins after embryo transfer, divided on morula stage, whereas one egg twins were obtained after transplantation of blastocysts, divided on all three stages of development.  And there are reports about possibility of using embryos of cows and pigs in the later stages of development for obtaining of one egg twins (Willadsen, 1981; Zambeth et al., 1983; Baker et al., 1984; Rorie et al., 1985). Effective test for assessing survival of embryo halves is cultivating them within 2-4 hours between separation and the transplant. Culturing embryo halves for a night or more than 12 hours, was accompanied by a marked decline in their survival.

  Chimeric animals. The concept of a chimera means a compound animal. In the modern concept of the term chimera is mainly used for obtaining composite organisms that have genetically different cell populations originate more than one zygote or more than one embryo. Obtaining genetic chimeras or mosaics is currently one of the promising areas of biotechnology. The essence of this biotechnological method, based on the achievements of cell engineering and micromanipulation on early embryo consists in artificial combining of cell embryos from two or more animals, relating not only to one breed, but also to the different breeds and even species. Chimeric animals are signs of different genotypes. This is achieved by integrating blastomeres from two or more embryos or by injection of cells of one embryo into the cavity of the blastocyst of another embryo. All previous known  mammalian chimeras were created by methods of aggregation of two (or more) of genotypically heterogeneous embryos or by microinjection of donors’ blastocyst intracellular cell mass in blastocoel of recipient. The first method is called aggregation, and the second is known as injection method Complex chimeric sheep embryos by integrating 2 -, 4 - and 8-cell blastomeres were obtained by Fehilly et al. (1984). Each of these embryos consisted of an equal number of blastomeres of embryos from 2-8 parents. Results of the survey of  48 lambs in 2  months of  age showed that 36 heads were  chimeric by blood tests, by external signs or in those and other indicators. A year later, Butter et al. got chimeric lambs by injecting inner cell mass isolated from donor embryos into  embryos’ blastocyst of recipients. From these 15 lambs 5 heads were identified as chimeras on blood groups and 1 head appears by its external signs. Chimeras in cattle were obtained by Brem et al. (1985) combining halves of 5-6-day-old embryos. 2 of 7 calves had evidence of chimerism. 1 calf was a chimera on suit of brown schwyz breed and Holstein-Friesian, although blood group it inherited from their Holstein-Friesian breed parents.  Another calf was uncertain chimera. Chimeric calves by merging morulae without zone of pellyutsida were obtained by Church et al. (1985). They found that the transfer of each parent type to chimera is random, ie, offspring can develop from cells derived from any embryo, or from a combination of embryos.
Obtaining chimeric by cell fusion of embryos from different species is of great practical importance. It is known  that no sheep or goat  nurture hybrid offspring before birth. Typically, the embryos of an experimental hybrid pregnancy in sheep and goats at the end of the 2nd month are killed. The immediate cause of abortion in interspecific pregnancy is the strengthening of maternal immune responses to antigens of the fetus, leading to dysfunction of the placenta. Fehilly et al. (1984) showed that the blastomeres of sheep and goats that are enclosed in agar and placed for 4-5 days into oviduct of  sheep  can form a combined blastocysts that are viable and can develop to birth normal offspring. The merger of one blastomere from 4-cell embryos of  sheep and goats 17 blastocysts were received, transplantation of  which was  ended with the birth of 7 lambs. They all looked like mostly in lambs, but 3 of them wool had that had  transverse ridges and patches of hair  sharply contrasting with the tight curly hair. By combining  8-cell embryos of sheep and goats researchers got five offspring similar to lamb, but 2 of them had similar deviations on coat, two offspring were like goat kids with some deviations into the coat. Animals with the outward signs of chimeras had blood sheep, except for one young animal, who had the blood group of parents of both species. The above experimental results indicate the feasibility of transplantation of chimeric embryos between closely related species of animals. Interspecies transplantation could be invaluable in preserving endangered species from extinction because usual embryo transfer may provide a small benefit, as the female recipient may not always be enough. Technique of obtaining  chimeras can be used in breeding animals with desirable economic characteristics, as well as resistant to certain diseases.
Chimeric animals do not transmit to  offspring their inherent genetic mosaicism. Like heterozygous or hybrid animals there is a splitting in the offspring, resulting in broken of valuable genetic combinations. Although chimeric animals support economic important signs  only for a single generation they can be of great practical interest in the breeding of cattle. For example, you can create chimeric animals that combine features such as milk and meat productivity, which are antagonistic and incompatible in a single body. Creation of chimeras by injecting of certain embryo cell lines will improve the immune system and increase resistance to a range of diseases.

 Cloning of animals is getting identical offspring by nuclear transfer of embryonic cells in germ cells with remote nucleus. Obtaining from high yielding donor cows five 32-cell blastomeres and transplantation each of nucleus into enucleated oocyt allows to receive from a donor 160 embryos simultaneously. Repeating this procedure with received "secondary" embryos that give opportunities for an unlimited number of offspring.

Nuclear transfer in amphibians was implemented by Briggs and King in 1952. They showed that the nuclei of early embryonic cells retain the ability to further develop after their transfer into enucleated oocyte. the Nucleus of specialized somatic cells of adult frogs, (ie passed deep differentiation), lost their totipotency (the property of cells to reach their full potential for the formation of a body), and in the best case, the development of genetic copies occurred to the early stages of the tadpoles. They also found that the nucleus of the embryos at a later stage of development, as well as less-developed embryos when transplanted into the oocyte do not split up. Newport and Korscher (1982) explained this by differentiation of embryonic cells, which appears at the stage when the embryo starts to produce its own RNA.

The first report of successful transplantation of mammals nuclei in mice appeared in 1981 (Illmense and Hoppe). In this experiment, the nucleus were extracted from the cells of the inner mass of the blastocyst and  with the help of a micropipette were transplanted into the zygote of  different lines of mice. Own pronucles of zygote was removed by the same pipette. After in vitro culturing  zygotes to the blastocyst stage embryos were transplanted to recipient - to mice of  third line.  Three obtained  offspring by genotype as well as by  phenotype were identical with the line of the donor mice. In 1986 Willadsen conducted nuclear transfer in sheep. He removed unfertilized oocyte in 30-33 hours after treatment with human chorionic hormone  at the beginning of hunting. Area of pellyutsida over ovum polar body was cut by fine glass needle. In an hour after holding ovum in the phosphate medium with cytochalasin polar body with the surrounding cytoplasm was aspirated. In such a way he succeeded to share about 90% of eggs in two parts with intact cell membranes, each containing about half of the cytoplasm. Cytological analysis showed that approximately 75% of those halves, which were removed from the polar body, contained a chromosome, ie, had "nuclear" halves of ovum, while the other half of the same oocytes did not contain nuclear structures, ie were enucleated. Single blastomeres of 8 - or 16-cell embryos were introduced under zone of pellyutsida of enucleated oocyte. Fusion of nucleus with the cytoplasm of the cell is accompanied by  Sendai virus or electric current. The cells included into agar and transplanted in sheep’s oviducts. After 4-5 days development of embryos were estimated. From 33% up to 48% of embryos reached blastocyst stage. In this experiment, transplantation of three from four blastocysts was ended  by the birth of the offspring. These lambs were obtained from embryos in which 8-cell blastomere  were transplanted into enucleated unfertilized oocyte. In another experiment pregnancy was achieved by embryo of 16-cell blastomeres.

Prather et al. (1987) transplanted blastomeres of two 32-cell  cow embryos into enucleated oocytes after maturation in vivo and in vitro by electrofusion. Oocytes extracted 36 hours after the start of hunting and used as recipients of the nuclei of embryonic cells more likely to achieve the stage of morula or blastocyst than ovum matured in vitro or extracted in 48 hours after the start of the hunt. 7 pregnancies achieved after transplantation of 19 embryos to 13 heifers. In 2 heifers calves were born alive. The above data suggest that the improvement of the efficiency of nuclear transfer technology of embryonic cells into enucleated oocytes allows to receive multiple copies of a single embryo.

Obtaining homozygous diploid offspring. At purebred line breeding is traditionally used,  the aim of which is to maintain a high genetic similarity with outstanding ancestor. This is achieved by moderate inbreeding and targeted selection. Animals such lines, with a high degree of homozygosity distinguished by the genetic similarity. Thus high phenotypic uniformity in respect of physiological and morphological traits is created in the line. Unfortunately, the creation of inbred animals requires a lot of time, because this is due to the splitting and recombination of genes, low fertility and a long interval between the generations.

The method of obtaining homozygous diploid offspring in many ways similar to the technology of nuclear transfer of somatic cells into enucleated zygote. But in the latter case heterozygous animals are obtained, but not homozygous for all genes of one parent. The method is as follows: from  zygote at the stage of two pronuclei male or female pronucleus is removed. As a result only one haploid set of chromosomes - male or female remains in the cell. For the development of the zygote, containing a haploid set of chromosomes, it is required to activate or restore the diploid set. To this a brief incubation of  haploid zygote in solution with cytochalasin B is carried out. The latter prevents the first cell division, but  division of nucleus and diploidization of the remaining pronucleus are activated. As soon as happened nuclear fission, the embryo is washed away from cytochalasin B   to prevent increase the number of chromosomes’ sets. 

The experiments showed that in mammals for the normal development of the embryo up to the birth both male and female pronuclei are necessary. It is believed that diploid genome derived from only one of the parents of the same sex can not ensure the normal development of the embryo. It is believed that the paternal genome is required for the formation of extraembryonic tissues, and maternal genome is necessary  to pass certain stages of embryogenesis. New data needed for theoretical understanding and experimental confirmation.  Long-term research  is necessary to overcome technical difficulties in obtaining homozygous diploid animals.

 

Definition and regulation of sex. Getting the animals of certain sex is not only biological, but also a practical problem. This is especially important for dairy cattle, whose main economic useful feature is milk production. And this feature refers to the attribute is bounded by  sex. Therefore there is very important task of regulating the sex ratio needed for effective breeding. Genetic mechanism of sex determination provides splitting offspring by sex in a 1:1 ratio. It is known that a set of homologous pair of sex chromosomes XX defines development of the female, and heterogeneous XY chromosomes determine development of the male. Over the years, investigations are underway to separate sperm carrying the X-and Y  sex chromosomes. For this purpose, various methods have been tested: centrifuge, sedimentation, electrophoresis, filtration, cytometry, immunoassays, etc. The most promising of them is the method based on the use of a laser. It is established that sperm with X chromosomes contain more DNA than sperm with Y-chromosomes. Thus the positive or negative charges of cells depends on the amount of DNA. First of all semen is processed by fluorescent dye, and then it is passed  through a laser beam. Under the influence of negatively and positively charged plates sperm is deviated to the appropriate direction. Currently this method is being introduced into practice  by «Sexing Technologies Navasota Texas». According to the manufacturer by means of this teehnologii it is possible to isolate fractions containing up to 92% of cells with X-or Y-chromosome. Since 2008  homosexual sperm is delivered to Kazakhstan. 

 

Test questions: 1. Tell us about the methods of obtaining identical twins proposed by researchers 2. What is the practical significance of chimeric animals (genetic mosaics)? 3. What are differences between the  method of obtaining homozygous diploid offspring and  animal cloning technology? 4. What method of sex regulating is being introduced into livestock of Kazakhstan?

 

Lecture №7


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