Achievements of Biotechnology in creating homozygous diploid offspring and improving technology of cloning animals
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.
Principles and approaches used in fertilization outside the body.
Egg maturation outside the body. Extracorporeal fertilization is preceded by the cultivation of oocytes in vitro, during which they mature to metaphase 2. Spontaneous resumption of meiosis of oocytes isolated from rabbit follicles and cultured in nutrient media was discovered in 1935 by Pinkus and Enzman. Moreover, the oocytes passed consistently all stages of maturation to metaphase 2, that is, to the stage of fertilization without any hormonal effects. Later this phenomenon was established in ovules of other animal species. By the time of the birth of the calf, its eggs reach the diplotent stage of meiosis prophase (a specific type of cell division), typical only for the sex cells. In this case, the formed sex cells contain a single (haploid) set of chromosomes. In the diplotent stage, homologous chromosomes diverge, a longitudinal slit forms between them. At this stage of meiosis, further maturation of the oocytes is suspended until the physiological maturity of the individual. An important role in the suppression of meiosis is played by granulosa cells and certain components of the follicular fluid. After raising the level of the luteinizing hormone in vivo or extracting the oocytes from the follicles, the inhibitory effect stops and the meiosis resumes. In this case, as noted above, the process of maturation of the egg to metaphase 2 continues, i.e. oocytes mature to a stage suitable for fertilization. However, it should be noted that this process is not equivalent to the maturation that the oocyte undergoes in vivo before its ovulation. Under conditions of cultivation of oocytes "in vitro", only the maturation of the nucleus occurs without the participation of the cytoplasm. Consequently, in vitro mature oocytes are not capable of further development after fertilization, since in this process the cytoplasmic factors responsible for the formation of the protein structure play an important role. Motlik, Fulka (1976) showed that during the normal development of the oocyte after fertilization an important role is played by substances released from the embryonic vesicle into the cytoplasm. Thibault et al. (1976) found that steroid hormones are required to ensure the complete physiological maturation of the oocyte. Moor and Trounson (1977) note that the cultivation of sheep eggs in medium 199 with the addition of follicle stimulating hormone (FSH) and luteinizing hormone (LH) compared to a medium without hormones ensures the metaphase reaches a large number of oocytes. Similar results were obtained with cows Fukui et al. (1982) using estradiol, progesterone, luteinizing and chorionic hormones. There was a marked effect of sex steroid hormones (estradiol, progesterone, testosterone) on the effectiveness of egg maturation, while the effect of gonadotropins (FSH, LH) was insignificant.
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Studies by Dielman et al. (1983) showed that during the maturation of oocytes, the content of sex steroid hormones in the follicular fluid of cows undergoes significant changes. Thus, in the first 6 hours after the release of LH in the follicular fluid, a high concentration of estradiol (a hormone produced by the ovary, adrenal gland, as well as the placenta and testes) is noted, then it sharply decreases and decreases by more than 10 times in comparison with the initial one by 20 hours. The testosterone content in the follicular fluid also undergoes a similar change. A more stable level during this period is noted in progesterone. The content of the latter was dramatically increased immediately before ovulation. These data prove the necessity of creating a non-static concentration of sex hormones in the culture medium during the maturation of eggs.
Moor, Trounson (1977) used the method of culturing them inside intact follicles to increase the usefulness of oocyte development. In their experiments, after transplantation of inseminated sheep with in vitro cultured oocytes in isolated follicles, up to blastocyst development occurred in 26-50% of the oocytes. Transplantation of blastocysts in 63% of cases resulted in the birth of lambs. Later it was found that many phenomena inside follicular cells, including steroid biosynthesis and protein synthesis, regulate gonadotropic hormones. Therefore, the latter should be an obligatory component of the medium in the cultivation of oocytes with follicular cells.
Staigmiller, Moor (1984), in order to elucidate the role of follicular cells in maturation, the oocyte cultivated them together without cumulus (cells on the surface of the shiny egg shell), oocytes with cumulus or oocytes with cumulus and follicular cells. Culturing was performed in medium TC 199 with 10% fetal fetal serum and gonadotropins (LH, FSH and prolactin) and estradiol. The oocytes were cultured at 37 ° C for 24 hours in a CO2 incubator with slow rocking, and then transplanted into the oviduct of the sheep in a hunt. The sperm was administered before transplanting the oocytes into both horns of the queens. After 10-12 days, the embryos were removed from the uterus and classified by category. The results showed that cultured in vitro oocytes without cumulus did not develop further. Adding follicular cells to the culture medium without cumulus did not improve the egg's ability to develop, as 83% of the oocytes remained at the unicellular stage. Oocytes with cumulus, but without follicular cells, remained unicellular in 87% of cases. Development before hatching blastocysts in 37% of oocytes was noted with the addition of follicular cells with cumulus. In another experiment, the cultivation of fertilized oocytes with cumulus and the layer of the granulation cells to be treated (cells of the young immature connective tissue provide the energy substrate to the oocyte) provided the development of 43% of the cells to the blastocyst, and after transplantation to their recipients embryonic survival reached 63%. The addition of follicular cells to this complex did not have a significant effect on the development of cells. The results of the studies indicate that for the development of fertilized oocytes a certain critical number of follicular cells is required, while their presence for the manifestation of meiosis is not necessary. Somatic cells serve not only as an energy substrate for oocytes, but also participate in the transfer of some precursors of amino acids, nucleotides and phospholipids to the oocyte. Follicular cells generate instructive signals that affect the nucleus and direct synthesis of certain structural proteins. These signals are very important for the maturation of oocytes in the first 6-8 hours after the initiation of meiosis. The change in signals leads to disturbances in the ontogenesis of the embryo. It is no coincidence that the method of culturing isolated follicles has been recognized by many researchers as the most modern method used to prepare oocytes for fertilization in vitro. This method makes it possible to preserve the natural connections between the oocyte and the somatic elements of the follicle. In this case, the follicle acts as a bio-plant of steroid hormones. The essence of the method is as follows. Follicles are isolated from the ovaries during the slaughter of cattle and delivered to the laboratory in the medium of TS-199. The ovaries are washed with a sterile physiological solution containing antibiotics, and then cut with a scalpel at the point of entry into it of the vessels. The follicles are clearly visible on the surface of the incision. Using tweezers, the follicles are separated from the connective tissue and cultivated in a CO2 incubator in the medium with the addition of 20% fetal fetal serum and the hormones of FFA, FSH, LH, estradiol and insulin for 48 hours. Under such conditions, approximately 70% of the oocytes mature to the stage of metaphase 2.
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In vitro fertilization. Fertilization of oocytes "in vitro" has now been achieved in more than 20 species of animals. In 1968, the first report appeared on the normal offspring obtained from mice, in 1974 - from rats, and in 1981-1984 - from cattle, pigs and sheep.
Fertilization of oocytes in vitro means that the complex physiological processes taking place in the body of the pregnant female should take place in relatively simple and static conditions. An important stage in the development of the method of in vitro fertilization was the discovery of the phenomenon of the capitation of spermatozoa Chang, Austin (1951). They established that fertilization only occurs if the spermatozoa are previously in the oviduct of a female for several hours before ovulation. At this time they undergo certain physiological changes and become capable of fertilization. It is believed that the sperm cellization is primarily a change or removal of protein and other macromolecular substances in the plasma membrane of the sperm. Proteolytic enzymes, necessary for penetrating the zone of the pellucida, are washed out through the plasma membrane. The duration of the capitation of mouse and hamster spermatozoa in the uterus and in vitro is 1-2 hours, rats 5-6 hours, while rabbits in the uterus about 6 hours, and in vitro for about 10 hours. Chang (1959) first obtained offspring after transplantation of ovules fertilized in vitro with spermatozoa, capated in the rabbit's uterus. After working out the conditions for the capitation of spermatozoa in the genital tract of the female, experiments were conducted on in vitro capation. For this purpose, oviductic and follicular fluids or blood serum were initially added to the medium. Later, in vitro fertilization and in vitro fertilization was achieved in mice in a medium containing bovine serum albumin and sodium pruvat without additives of biological fluids. The main medium for capping spermatozoa and in vitro fertilization in rats and hamster mice was a Krebs-Ringer solution containing glucose, serum albumin, lactate and sodium pyruvate.
Oliphant, Brackett (1973), using immunological methods, showed that the process of capation involves removing or rearranging the components of the seminal plasma that cover the surface of spermatozoa. The same researchers proposed a method of capping spermatozoa in vitro, which consisted of the following. The spermatozoa were washed from the seminal plasma by suspension in a definitive isotonic or hypertonic medium and centrifuged. The supernatant was drained and the pellet was resuspended in fresh medium. They were then incubated in the medium for 20 minutes, after which they were subjected to centrifugation at room temperature. Subsequently, the spermatozoa were incubated in a water bath at 38 ° C. In vitro fertilization was carried out as follows. Oocytes with cumulus cells were collected from the ovary surface and placed in small petri dishes containing the definitive medium. Fertilization was performed by adding approximately 1 million spermatozoa to a cup containing freshly ovulated eggs, after which gametes were incubated in a humid chamber with 5% CO2 in air at 38 ° C. The researchers transplanted 176 two- and four-cell embryos. Of these, 24 embryos, or 13.1%, were implanted, but most of them were resorbed.
In further experiments, the optimal concentration of spermatozoa was determined for in vitro fertilization of oocytes from laboratory animals. It has been established that when in vitro fertilization the sperm heads penetrate the pellucida zone in hamsters for 3-7 minutes. In mice and rats, the duration of penetration of the capated spermatozoa through the zone of the pellucida and the transformation of the spermatozoon head into the male pronucleus is approximately 2 hours, and the fragmentation of the fertilized egg occurs 30 hours later.
LK Ernst and co-authors. (1983) carried out in vitro fertilization without pretreatment of spermatozoa. In these experiments, the bull sperm were washed twice in Brinster's or TC 199's. The authors conclude that the main point in fertilization is the full maturation of the oocytes. The percentage of crushing oocytes was the same when the mature eggs were inseminated with capated and non-capated spermatozoa. According to the researchers, the sperm cells are a natural process that ends when passing through the cumulus cells near the ovum pellucid zone. If the egg is full and surrounded by cumulus cells, then the sperm cells are capitated normally at the time of their contact with the zone of the pellucida.
The results achieved in laboratory animals enabled scientists in the early 1970s to begin developing a method of in vitro fertilization in domestic animals. It should be noted that, if most of the experimental studies were performed on oocytes after maturation in vivo, then similar experiments with agricultural chemicals. animals were carried out mainly on eggs, previously cultivated in vitro to ripen them.
Sreenan (1970) and Trounson et al. (1977) did not observe the penetration of spermatozoa into the ova of cows transplanted into the rabbit's oviducts, where spermatozoa of the bull were introduced. The result was positive when used as an incubation system for oocyte oviducts oocytes. Iritani, Niwa (1977) fertilized oocytes of cows in vitro with spermatozoa aged 12-14 hours in the oviduct of the estrous rabbit. The same researchers conducted experiments on the capitation of bull spermatozoa in the isolated sexual tract of the estrous cow. The sperm was placed in an isolated genital tract of the cow (horn of the uterus and oviduct), after washing it with Krebs-Ringer's solution. After the introduction of spermatozoa, the ovarian end of the oviduct, the uterine tube joint and the cervical canal of the uterus were ligated and the genital tract was placed in saline for 3-4 hours at 37 ° C. Then the oviducts and uterine horns were washed to extract spermatozoa, and the latter were introduced into the medium with cultured eggs for 18-21 hours. The fertilization of eggs with the formation of two pronuclei was achieved when using spermatozoa, both in the oviduct (7%) and and in the horn of the uterus (6%). Further improvement of the described technique allowed Iritani in 1980 to achieve the fertilization of 22-26% of the oocytes. These eggs were crushed to the 2-4-cell stage under in vitro and in vivo conditions. In these experiments, it was established that for the spermatozoa of a bull in the genital tract of cows or females of another species, 4 to 6 hours are required.
Fertilization of oocytes of cows with spematozoids, encapsulated in vitro, was achieved in the late 70's. For this purpose, Bracett et al. (1978) used a physiological medium with high ionic strength. In these experiments, unlike previous experiments in which oocytes were used after ripening in vitro, the oocytes were removed from the pre-follicular follicles or from the oviducts shortly after ovulation. According to the researchers, 14 of the 25 eggs were fertilized, and 10 of them reached the 2-cell stage in 24 hours and 4 out of 7 oocytes - the 4-cell stage in 48 hours. Later, Iritani et al. (1984) proved that spermatozoa can be capacitated not only in a medium with a high ionic strength, but also in an isotonic medium. Moreover, the authors came to the conclusion that the capa- cation is possible during the storage of the spermatozoon at a temperature of 20 ° C.
The in vitro fertilization efficiency is largely dependent on the factors associated with the eggs (Brackett et al., 1982). Thus, the penetration of spermatozoa, encapsulated in a medium with a high ionic strength, was registered in oocytes in 40% of ovules that matured in pre-ovulatory follicles or oviducts (so-called tibial oocytes), whereas in oocytes ripened in vitro, only 10% were fertilized. Later, the effectiveness of this experiment was confirmed by the authors in obtaining twins. 2 out of 5 surgical transplants culminated in double pregnancies. However, the fertilization efficiency was low -11-14%. Embryos under in vitro cultivation conditions developed up to the 4-8-cell stage, which prevented the experimenters from performing non-surgical transplantation (for non-surgical transplantation and freezing, embryos need to be brought to the stage of compaction). However, even if a perfect technique for extracorporeal fertilization of tibial oocytes is developed, the huge genetic material of the oocytes contained in the follicles will remain unrealized in reproduction and breeding. Only the development of the method for obtaining calves from extracorporeal fertilization of follicular oocytes (ovules matured in vitro) will create a real possibility of a significant increase in the efficiency of transplantation and the creation of large embryo banks from genetically valuable cows. Such a technique will also ensure cheaper production of a sufficient number of zygotes and early embryos, necessary for the transfer of nuclei and recombinant DNA, as well as for the production of transgenic animals.
The first calf from the ripe "in-vitro" follicular oocyte after its in vitro fertilization was born in 1983 (LK Ernst et al.). Researchers for this purpose used oocytes matured in vitro, and uncapacitated fresh or frozen-thawed spermatozoa. Each recipient-heifer was surgically transplanted into the oviduct with 1-7 embryos located in the 2-4-cell stage of crushing. One recipient was transplanted 3 4 cell embryos obtained from in vitro extracorporeal fertilization of mature follicular oocytes taken from the ovaries of two- and one-month-old calves and one mature cow. As a result of such transplantation, a live calf was born. However, surgical transplantation of embryos located in the first stages of development into the recipient's oviduct significantly complicates biotechnology of transplantation. Therefore, it is very important to obtain embryos at the stages of the morula or blastocyst that are suitable for transplantation in a non-surgical way.
LK Ernst and co-authors. (1987) used the rabbit's oviduct to provide early stages in the development of cow embryos both after fertilization in vitro and after fertilization in the rabbit's oviduct (1987). They extracted the oocytes from the follicles and cultured in medium 199 with 20% fetal fetal serum with the addition of estradiol, progesterone, testosterone and luteinizing hormone for 24 hours at 38 ° C in a humid chamber containing 5% CO2, 5% O2 and 90% N2. The spermatozoa were capped in a medium with high ionic strength and in the oviduct of the estrous rabbit for 4-5 h before oocyte transplantation. 19-20 hours after the connection of the ovules with spermatozoa in the rabbit oviduct or in vitro, the oocytes were transplanted into the ligated oviduct of the pseudopregnant rabbit for their further development for 4-5 days. Fertility was 22-25%. After 3 days, the percentage of embryos reaching the stage of morula after fertilization in the rabbit oviduct is approximately 4 times higher than that after fertilization in vitro. On days 4 and 5, this difference decreases somewhat, but still remains significant in favor of oocytes fertilized in the rabbit's oviduct. This indicates that in the rabbit's oviduct, more favorable conditions are created for the capitation of the bull's spermatozoa and the fertilization of the oocytes than in culture cultures known so far. As a result of surgical and non-surgical transplantation of cows' embryos in the morula stage, 6 pregnancies were obtained, including one double (5 out of 6 pregnancies were given by oocytes taken from the ovaries of cows killed at the meat-packing plant). Two live calves were born from oocytes fertilized in the rabbit's oviduct and "in vitro". One calf was born as a result of non-surgical transplantation of an embryo obtained after in vitro fertilization. The implantability of embryos obtained as a result of fertilization of oocytes in the rabbit oviduct was almost 6 times higher than in cases of fertilization "in vitro".
For the development of bovine embryos prior to the morula stage and blastocysts, Crister et al. (1986) used ligated sheep oviducts. Significant success in fertilization in vitro of cow oocytes was also achieved by Irish scientists (Lu et al., 1987, 1988). As a temporary recipient for fertilized cells, they also used the oviduct of the sheep. 80% of the oocytes were broken up to the 2-cell stage and above, and almost half of them reached the stage of morula and blastocyst.
Experiments on the fertilization of sheep were carried out in two directions: "in vitro" and the introduction of follicular oocytes into the oviduct of the inseminated sheep. As in cows, the efficiency of oocyte fertilization was higher when follicular and ovulated cells were placed in the oviduct with spermatozoa than with the in vitro system. Moreover, the researchers did not observe a significant difference in the development of oocytes before the blastocyst stage in cases of egg culture in vitro and in vivo (in follicles). To a lesser extent the problems of fertilization "in vitro" in pigs have been studied. Polge (1977) described the penetration of spermatozoa into oocytes after maturing them in vitro and transplanting into the oviduct of an inseminated estrous pig. However, none of the eggs developed to the stage of blastocysts, and many of them developed polyspermy.
Cultivation in vitro of embryos of farm animals. Species serum was the first medium that was tested for suitability for the cultivation of cows and sheep embryos. In this environment, the development of fertilized cells was limited to one division and ceased after 48 hours of cultivation.
The possibility of using a follicular fluid for embryo culture was reported by Thibault in 1966. He observed in this environment the development of the embryo of cows from 1 to 4 cell blastomeres to the morula. Tervit et al. (1972) experienced a synthetic oviductive fluid (SNJ). According to the authors, 9% of the 8-cell blastomeres of sheep developed before the early blastocyst stage after 6 days of cultivation in the CLJ in an atmosphere consisting of 5% CO2, 5% O2, and 90% N2. More than half of the 8-cell embryos cultured in the CLJ for 3 days after the transplantation passed the ontogenesis path before the offspring. However, the authors were unable to obtain pregnancies after the cultivation of unicellular embryos. Bowan et al. (1975) noted the predominant development of embryos in the media of the CLL and XEM F-10 with an osmotic pressure of 270 mOsm compared to 300 mOsm. The latter medium with the addition of fetal serum, according to a number of scientists, is the most favorable environment for the cultivation of embryos "in vitro." For example, Wright et al. in 1976 in the environment of CHEM F-10 observed the development of embryos of cows from 1-2 cell blastomeres to hatch blastocysts. The following year, Peters et al. similar results were achieved using Witten's medium supplemented with 0.5% bovine serum albumin. They also noted that "in vitro" the development of 1-4-cell embryos is limited compared to the 8-cell embryos. This medium, but with an increased content of bovine serum albumin (15%), was optimal for the cultivation of pig embryos (Linder, Wright, 1978).
At present, scientific research in the field of fertilization of agricultural workers.animals "in vitro" receive further development. They are aimed at excluding from this technology a temporary recipient (oviduct rabbit, sheep, etc.) for the development of early embryos. A suitable replacement for the time recipient may be a monolayer of the oviductive and granulation cells.
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