Method for obtaining unisexual female offspring in sturgeon fish

FIELD: genomic engineering, pisciculture.

SUBSTANCE: the present innovation deals with induction of embryonic gynogenetic development due to inseminating ovicells with genetically inactivated sperm. Moreover, induction should be applied for ovicells of the females in the hybrids of phylogenetically distant species. Such females are able to produce unreduced diploid ovicells which are genetically identical to maternal ones. The innovation enables to create clonally reproducing female lines of sturgeon fish, (unisexual female offspring) in fish with different mechanisms of sex detection, the case when the mechanism is unknown, among them. Also, it gives the chance to considerably reduce expenses at forming a spawning school.

EFFECT: higher efficiency.

1 ex

The invention relates to genomic engineering in the field of fish farming and, in particular, is aimed at addressing the regulation of sex in sturgeon, producers of valuable food product is black caviar.

In a sharp reduction in natural populations of sturgeon modern sturgeon breeding mainly focused on the obtaining of eggs and offspring for their commercial implementation. Late maturation of sturgeon fishes (7-15 years) and the inability to determine the sex until the age of puberty entails the overhead of nursery areas, feed, energy and labor, since males make up about 50% of the herd. In this regard, it is highly important to obtain same-sex female offspring sturgeon, which can solve the problem of mass production of sturgeon caviar.

There are two approaches through which you can adjust the floor in fish: hormonal regulation and genetic regulation (Kirpichnikov V.S. Genetics and breeding of fish. Leningrad: Nauka, 1987, 520 S.). The first approach consists in changing sex composition in the offspring of fish by exposure to sex hormones. The second approach involves manipulation of the genomes of fish, including the method of induced gynogenesis.

A method of obtaining a same-sex female offspring sturgeon by hormonal regulation of sex (Omot H, Maebayashi M., Mitsuhashi E., Yoshitomi, K., Adachi Sh., Yamauchi K. Effects of estradiol-17β and 17α-methyltestosterone on gonadal sex differentiation in the F₂hybrid sturgeon, the bester // Fisheries Sciences. 2002. Vol.68. No 5. P.1047-1054). The method consists in the fact that in a certain period of development on fish effect of female sex hormones - estrogens. This action leads to feminization of males, i.e. the development of testes instead of the female gonads - ovaries. In the known method of juveniles Baster (hybrids between Beluga, Huso huso and the sterlet, Acipenser ruthenus) were fed the feed containing the female sex hormone estradiol. The resulting offspring that 95% consisted of females. The disadvantages of the method are: the impossibility of receiving the inversion of the sex of all individuals; the suppression of reproductive function in genotypic females and the occurrence of male intersex (hermaphrodites) under the influence of hormones; in addition, the impact must be each new generation of fish. In this regard, a more promising direction of genetic regulation of sex.

The closest analogue of the claimed invention is a method of obtaining a same-sex female offspring of the American paddlefish, Polyodon spatula, belonging to the family Velenosi, detachment sturgeon-like (S. Mims Aquaculture of paddlefish in the United States // Aquat. Living Resour. 2001. Vol 14. P.391-398).

The known method includes the ri phase: induction of gynogenetic development of embryos by fertilization genetically inactivated sperm, hormonal sex inversion obtained gynogenetic females and breeding males of invercantv with normal females to obtain same-sex female offspring.

The induction of gynogenetic embryo development in paddlefish was performed using activation of oocytes UV-irradiated heterologous sperm followed by diploidization embryos thermal shock, which facilitated the return of the second dialects of Taurus. Then gynogenetic individuals aged 20 weeks were implanted prunaprismia capsules containing methyltestosterone. All of gynogenetic individuals not subjected to hormonal effects, were females. About 90% of the genotypic females were inverted in males. Now grown offspring from crosses of males invercantv with normal females. There is every reason to believe that this offspring will consist only of females.

This method of obtaining same-sex female offspring can be implemented only in species with female googletest (genotype females have two identical sex chromosomes - XX) and male heterogametes (genotype males have two distinct sex chromosomes - X and Y).

The mechanism for obtaining same-sex female offspring known method consists in the following.

Induced genagent is a method to the torus get offspring, development which is carried out only at the expense of all the chromosomes of the egg. For exceptions to development of the male set of chromosomes of the sperm subjected to genetic inactivation using ionizing or ultraviolet radiation. Irradiated sperm only activate egg development, but their chromosome sets in the development of gynogenetic embryos are not involved.

Gynogenetic embryos have a haploid set of chromosomes is reduced trunk in the process of meiosis, the number of autosomes and one X chromosome. Haploids in fish cannot develop normally and die during embryogenesis or shortly after hatching, non-feeding larvae. Diploidization gynogenetic haploids carried out with the help of suppression of the second meiotic division, exposing young embryos thermal shock, which causes the Union of haploid chromosome sets of the egg and the second referring Taurus. This restores the diploid set of autosomes and sex chromosomes. Because when women googletest sex chromosomes are the same, all of gynogenetic diploid individuals have the same set of sex chromosomes - XX. Thus, in species with female googletest diploid gynogenetic offspring represented only by females (XX). However, when diploidization by combining two subsidiaries of the sets of chromosomes almost all the genes of gynogenetic individuals moving in the homozygous state, which is manifested in a significant decrease in viability and fertility of gynogenetic fish (inbreeding depression). Many females are completely sterile and fertile produce a small amount of caviar. For ROE fishery gynogenetic females unusable. So gynogenetic offspring, consisting of females use to turn into males, exposing the effects of male sex hormones, androgens. When this occurs masculinize females instead of ovaries develop testes. The difference between these male-invercantv XX from normal male XY is that all produced their sperm have only sex chromosome X. Males-invercantv XX used for crossing with normal XX females and get offspring, represented only by females. Same-sex female offspring obtained in this case has already normal levels of heterozygosity and hence normal viability and fertility.

The disadvantages of this method should include its complexity and the need to apply to embryos and fish the many influences (temperature shock, hormonal treatment).

A significant limitation on this method imposes the fact that it applies only to species of fish with female googletest. Meanwhile floor many who Idov fish is determined by another mechanism. Females of such species are not the same and different sex chromosomes (W and Z), and males on the contrary, the same (ZZ). Female heterogametes WZ leads to diploid gynogenetic offspring consists of fish with different sexual genotypes: WZ - female, ZZ males and WW. The sex of the fish with genotype WW while not known for sure. The heterogeneity of the sex composition of gynogenetic offspring is not possible to use it to get same-sex female offspring in a known manner.

American paddlefish with female googletest, belongs to the family Polyodontidae, and all the rest of sturgeon fish - producers of black caviar (Beluga, sturgeon, sturgeon) of the family Acipenseridae. It is shown that one of the representatives of the family Acipenseridae, white sturgeon (Acipenser transmontanus), has a female heterogametes (Van Eenennaam A.L. Van Eenennaam JP, Medrano J.F., Doroshov, S.I. Evidence of female hetero-gametic genetic sex determination in white sturgeon // J. Hered. 1999b. V.90. P.231-233). The mechanism of sex determination in other species of sturgeon have not been studied.

The technical result of the invention is to create a clonal breeding lines of the female sturgeon that allows you to get same-sex female offspring of fish with any mechanism of sex determination, including when the mechanism is unknown, and gives an opportunity to significantly reduce costs in the formation of breeding herds.

The phenomenon of narducci eggs are known in many species (natural forms) fish (Vasil'ev V.P. Evolutionary caryologia fish. M.: Nauka, 1985, 301 S.). All these forms are of hybrid origin. Multiply these forms with natural gynogenesis, i.e. their eggs have evolved special cytoplasmic mechanism that eliminates the male set of chromosomes from development. Deploymnet oocytes leads to normal development of gynogenetic individuals.

Deploymnet of oocytes from natural hybrids and their gynogenetic offspring due to the special transformation of meiosis, which is that even before the first meiotic division in the chromosomes flows additional DNA replication (endoreduplication), resulting in chromosome number is doubled. In this regard, in the first division of meiosis conjugat the I and recombination occur not between homologous, and genetically identical sister chromosomes. In addition diplodonta genetic consequence of this transformation of meiosis is that all ripened egg isogeny and identical to the genotype of the mother. They differ from ordinary fish, meiotic recombination which leads to genetic diversity (variability) produced eggs. The phenomenon of narducci egg does not occur in any of the hybrids, and only if certain (not too big and not too small) phylogenetic distance of the species that comprise a hybrid combination.

Usagenote eggs, their identity maternal genotype and their mechanism of inactivation of chromosomal sperm (natural genagent) leads to the fact that all individuals developing from these eggs are a kind of clone of reproducing the genotype of the mother, i.e. clonal inheritance. Therefore, this clone consists only of females and is, therefore, same-sex female. Sex composition of offspring in this case does not depend on the female Homo - or heterogametes.

Preliminary research has shown that some artificial hybrids phylogenetically distant species of sturgeon are also capable of similar transformation of meiosis, in which the hybrid females with great is th or less frequently produce deregulirovanye diploid eggs genetically identical to the mother. Therefore, in the present method of obtaining same-sex female offspring sturgeon induction of gynogenetic development applies to the eggs of the female hybrids phylogenetically distant species. When such insemination of oocytes genetically inactivated (irradiated) sperm resulting offspring is an identical mother clone, and therefore, all fish in the offspring are females.

Thus, the inventive method allows to obtain clonal breeding lines females sturgeon regardless of the mechanism of sex determination. This enables the formation of the brood stock, consisting of some females, with the aim of further mass production of sturgeon caviar. This eliminates the need for the identification of sex, apply to embryos and fish such effects as thermal shock, hormonal treatment, and reduced the cost of cultivation of males. Receive clonal breeding lines females has the advantage that, for the cloning of the first generation hybrids of all subsequent generations of genetically identical to the first, keeping its fisheries indicators, whereas normally sturgeon hybrids the most productive in the first generation, and subsequent generations, their productivity decreases. In addition, when the COI is whether the proposed method solves the problem of Biosafety because in case of contact with clonal hybrid females in natural waters and their crosses with loved ones pure offspring will be triploid and therefore sterile.

Thus, the set of distinctive features ensures the achievement of the technical result.

In the analysis of the prior art is not detected analogues characterized by signs, identical with all the essential features of the claimed invention. Therefore, the claimed invention meets the condition of "novelty."

A new set of features ensures the achievement of a new technical result and does not follow for the expert in the obvious way from the prior art as never before in sturgeon and generally in vertebrates has not been artificially obtained same-sex female offspring by cloning females with unknown mechanism of sex determination. Thus, the claimed invention meets the condition of "inventive step".

Information confirming the possibility of carrying out the invention with obtaining the specified technical result.

The method is as follows.

Get hybrids sturgeon phylogenetically distant species, females are capable of producing diploid deregulirovanye aiticle the key. When this is chosen such hybrid females, the eggs are larger than normal (diploid oocytes larger than haploid), or consists of a mixture of large (diploid) and small (haploid) eggs. Is irradiated with the added one of the parental species or male hybrid ionizing or UV radiation in the dose that causes a complete genetic inactivation of all sperm. Inseminated oocytes hybrids irradiated sperm. When this happens induction of gynogenetic embryo development. Applying thermal shock to diploidization of gynogenetic haploid embryos not required, as the egg, which occurred dramaticheskaja chromosome endoreduplication already diploidy. The vast majority of gynogenetic embryos developing from reduced egg will remain haploids and die. Thus, the output of viable gynogenetic larvae can judge the frequency of endoreduplication in this hybrid combination, and this hybrid females.

Diploid viable gynogenetic larvae can occur in two ways: due to spontaneous suppression of the second meiotic division or by diplodonta eggs due to endoreduplication of DNA. It is proved that the frequency of occurrence of larvae due to spontaneous suppression of the second meiotic division sturgeons market is very small and amounts to about 0.1% (Recoubratsky AV, Grunina A.S., Barminas VA, Golovanov T.S., Chudinov O.S, Abramova A.B., Panchenko NS, Kupchenko S.A. Meiotic genagent the sturgeon, Russian sturgeon and sterlet // Ontogeny. 2003. 32. No. 2. S-131). These larvae will not isogenic, and may differ from females on individual genetic characteristics. They can develop both in females and males. Larvae that emerged due to diplodonta eggs due to endoreduplication of DNA, are a clone are genetically identical to the mother. Of them can develop only females.

Growing and gynogenetic reproduction of hybrid females are capable of high frequency to produce diploid eggs, allows you to generate the brood stock, from which it will be possible on an industrial scale to obtain same-sex female offspring sturgeon.

An example of the method.

The method implemented in the hybrids between Beluga and sterlet (hybrid Bester). Battery received for a long time and are used in aquaculture. The choice of this hybrid sturgeon made on the basis of data on fertility, fecundity, and size of the eggs of Nesterov, which suggests that some of the eggs produced by females Baster, is diploid, meaning that their chromosomes are formed in dramaticheskoi andrewblake the AI chromosomes.

From 15 females Baster, 5 females starlet and 2 females Beluga got caviar. Sperm was obtained from 5 male Baster and mixed it. From eggs obtained from each female were selected to equal portions, which were used in the experiment for making crosses. All males and females after receipt of gametes were taken and fixed in 96% ethanol fragments fin for subsequent molecular genetic analysis. Part of the sperm taught shortwave UV light for 7 minutes Prior research has shown that this procedure causes a full genetic inactivation of sperm, however, the added after irradiation retain mobility and are able to activate the egg to gynogenetic development.

Using eggs from each female were placed the following crosses:

(1) female Baster × male Baster insemination normal sperm (control);

(2) female Baster × male Baster insemination irradiated sperm;

(3) female sterlet × male Baster, normal sperm (control);

(4) female sterlet × male Baster insemination irradiated sperm;

(5) female sterlet × male Baster insemination irradiated sperm, heat shock;

(6) female Beluga × male Baster insemination normal sperm (control);

(7) female Beluga × male Baster, EfE is the change irradiated sperm.

(8) female Beluga × male Baster, insemination irradiated sperm, heat shock.

Embryos were incubated in Petri dishes at a temperature of 20°C. in 5-10 min after insemination, embryos from crosses 5 and 8 were subjected to heat shock at a temperature of 37°With a duration of 2.5 minutes Heat shock at this point, suppresses development in embryos second division of meiosis, thereby restoring deploymnet. Incubation was continued for 7 days. The non-feeding larvae hatching lasted for 2 days. The larvae transition to active feeding occurred in 6-9 days after hatching.

In the crosses obtained the following results.

All crossings of the control options (1, 3, 6) received normal prelicense (exit from the number of fertilized eggs from 65 to 85%), of whom most (75-90%) and further developed normally and proceeded to active feeding.

Almost all embryos from crosses in which caviar sterlet and Beluga were seminal irradiated sperm (options 4 and 7), developed abnormally, they had clear signs of haploid syndrome (curvature of the axial skeleton, the hydration of the pericardium, filiform heart). All embryos from crosses of 7 died before hatching. Most embryos from option 4 also died before hatching. Just hatched among non-feeding larvae (0-2% in different crosses) found tol is to 4 morphologically normal instance (0.005%). All ugly prelicense died within a few days after hatching, before the transition to active feeding. Two of the four normal larvae began to active feeding. They can occur due to spontaneous suppression of the second meiotic division in the fertilized ovum, which led to the restoration of the diploid status of gynogenetic embryos.

In versions 5 and 8, in which for diploidization of gynogenetic haploids sterlet and Beluga with suppression of the second meiotic division was used heat shock, were obtained from normal diploid larvae (10-15%).

In crosses of option 2, in which irradiated sperm osumenyi spawn Bester, marked by a large number of normally developing embryos. The output of morphologically normal non-feeding larvae in different crosses (eggs of different females) ranged from 3%to 25%. Most of them (65-85%) started active feeding.

Experienced (173 units, option 2) and control (50 pcs., option 1) Nesterov were grown in aquariums. In addition, grew to 30 pieces of diploid gynogenetic individuals sterlet (option 5) and Beluga (option 8). At the age of 2 months with all fish were taken fragments of the fins for in vivo molecular-genetic analysis. The DNA isolation from the fins of juveniles and their parents was performed by standard method. Researched RAPD-pectra total DNA polymorphism of microsatellite sequences (4 loci).

The analysis showed that DNA control fish (option 1) contain alleles of microsatellite loci available as mothers and fathers, and their RAPD-spectra consisted of a combination of the bands characteristic of both parents. Thus, in this case there was a picture, which takes place under normal crossing.

The frequency of homozygotes microsatellite loci among gynogenetic individuals sterlet and Beluga (options 5 and 8) was significantly higher than in control. Gynogenetic individuals differ among themselves, and also differed from mothers of both sets of microsatellite alleles and RAPD-spectra. Genetic differences in gynogenetic progeny of the pure species caused by divergence in different sex cells of homologous chromosomes and recombination of chromosomes during meiosis.

In the experimental apparatus (2) most individuals (80 to 90%) identical investigated indicators and not different from their mothers, i.e. they can be identified as the parent clones. This situation could arise only in the case when some of the eggs of females Baster was diploid due to endoreduplication DNA in early meiosis. All of these fish can only be females. Identified genetic differences among small part experienced fish can be caused by changes in endoreduplication DNA, as well as mutate the mi. Females Baster, which has the highest output of gynogenetic diploid non-feeding larvae, were selected for further reproduction to form a brood stock, from which it will be possible on an industrial scale to obtain same-sex female offspring sturgeon.

Thus, the inventive method allows to obtain clonal breeding lines females sturgeon (same-sex female offspring), regardless of the mechanism of sex determination, thereby reducing material costs during the formation of the escapement. Similarly, same-sex female offspring can be obtained not only Bester, but hybrids between other species of sturgeon, provided that these hybrids are able to produce deregulirovanye gametes that are genetically identical to the maternal genotype.

The above information suggests that the method of obtaining a same-sex female offspring of sturgeons in the claimed invention relates to genomic engineering in the field of fish farming and, in particular, aimed at solving the problems of the regulation of sex in sturgeon. For the inventive method, it is described in the above-mentioned claims, confirmed the possibility of its implementation using the steps described in the application of tools and techniques. Therefore, the claimed invention shall correspond to the condition of "industrial applicability".

A method of obtaining a same-sex female offspring of sturgeon, including the induction of gynogenetic development of embryos by fertilization genetically inactivated sperm, characterized in that the induction of gynogenetic development applies to the eggs of the female hybrids phylogenetically distant species, capable of producing deregulirovanye diploid egg cells, genetically identical to the mother.