RU2312495C2 - Method for obtaining unisexual female offspring in sturgeon fish - Google Patents

Abstract

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

Description

The invention relates to genomic engineering in the field of fish farming and, in particular, is aimed at solving the problem of sex regulation in sturgeons, producers of a valuable food product - black caviar.

In the conditions of a sharp reduction in the natural populations of sturgeon fish, modern sturgeon breeding is mainly focused on obtaining eggs and offspring with a view to their commercial sale. Late maturity of sturgeon fish (7-15 years) and the inability to determine sex up to the age of puberty entail unproductive costs of growing areas, feed, energy and labor, because males make up about 50% of the herd. In this regard, it is very important to obtain homosexual offspring from sturgeons, which allows to solve the problem of mass production of sturgeon caviar.

Two approaches are known that can be used to control the sex of fish: hormonal regulation and genetic regulation (B. Kirpichnikov, Genetics and fish breeding. L .: Nauka, 1987, 520 pp.). The first approach is to change the sexual composition in the offspring of fish by exposing them to sex hormones. The second approach involves manipulating fish genomes, including the method of induced gynogenesis.

There is a method of producing same-sex offspring in sturgeon by hormonal regulation of sex (Omoto H, Maebayashi M., Mitsuhashi E., Yoshitomi K., Adachi Sh., Yamauchi K. Effects of estradiol-17β and 17α-methyltestosterone on gonadal sex differentiation in the F ₂ hybrid sturgeon, the bester // Fisheries Sciences. 2002. Vol. 68. No. 5. P.1047-1054). The method consists in the fact that at a certain period of development, fish are affected by female sex hormones - estrogens. Such an effect leads to the feminization of males, i.e. the development of the ovaries instead of the testes of the female sex glands. In a known method, juvenile bester (hybrids between beluga, Huso huso and sterlet, Acipenser ruthenus) fed food containing the female sex hormone estradiol. As a result, offspring were obtained, which 95% consisted of females. The disadvantages of the method are: the inability to obtain sex inversion in all individuals; inhibition of reproductive function in genotypic females and the emergence of male intersex (hermaphrodites) under the influence of hormones; In addition, each new generation of fish must be exposed. In this regard, the direction of genetic regulation of sex seems more promising.

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

The known method includes three stages: induction of the gynogenetic development of embryos by fertilizing the eggs with genetically inactivated sperm, hormonal inversion of the sex of the obtained gynogenetic females and mating of male inverters with ordinary females to obtain homosexual offspring.

Induction of the gynogenetic development of embryos in the paddlefish was carried out by activating the eggs with UV-irradiated heterologous sperm, followed by diploidization of the embryos by heat shock, which facilitated the return of the second guide body. Then, semi-impermeable capsules containing methyltestosterone were implanted to gynogenetic individuals at the age of 20 weeks. All gynogenetic individuals not subjected to hormonal effects turned out to be females. About 90% of genotypic females were inverted to males. Offspring are currently being raised from crossbreeding males with normal females. There is every reason to believe that this offspring will consist only of females.

This method of obtaining homosexual progeny can be implemented only in fish species with female homogameticity (in the female genotype there are two identical sex chromosomes - XX) and male heterogameticity (in the male genotype there are two different sex chromosomes - X and Y).

The mechanism for obtaining same-sex offspring in a known manner is as follows.

Induced gynogenesis is a method in which offspring are obtained, the development of which is carried out only due to the chromosome set of an egg. To exclude the development of the male set of chromosomes, sperm are genetically inactivated by ionizing or ultraviolet radiation. Irradiated sperm cells only activate the eggs to development, but their chromosome sets do not participate in the development of gynogenetic embryos.

Gynogenetic embryos have a haploid set of chromosomes - the number of autosomes reduced during meiosis and one X chromosome. Haploids in fish cannot develop normally and die during embryogenesis or shortly after hatching of larvae. Diploidization of gynogenetic haploids is carried out by suppressing the second division of meiosis, exposing the early embryos to temperature shock, which causes the union of the haploid chromosome sets of the egg and the second guide body. In this case, the diploid set of autosomes and sex chromosomes is restored. Since sex chromosomes are the same with female homogametism, all gynogenetic diploid individuals have the same set of sex chromosomes - XX. Thus, in fish species with female homogameticity, diploid gynogenetic offspring are represented only by females (XX). However, during diploidization due to the combination of two daughter sets of chromosomes, almost all genes of gynogenetic individuals turn into a homozygous state, which is manifested in a significant decrease in the viability and fecundity of gynogenetic fish (inbred depression). Many females are completely sterile, and fertile ones produce a small amount of eggs. Gynogenetic females are unsuitable for caviar fish farming. Therefore, gynogenetic offspring, consisting of females, are used to turn them into males, exposing them to male sex hormones, androgens. In this case, masculinization of females occurs - they develop testes instead of ovaries. The difference between such male inverters XX from ordinary males XY is that all sperm produced by them have only the sex chromosome X. Male inverters XX are used for interbreeding with ordinary females XX and produce offspring represented only by females. The same-sex offspring obtained in this case already have a normal level of heterozygosity and, therefore, normal vitality and fecundity.

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

A significant limitation on this method is imposed by the fact that it is applicable only to fish species having female homogameticity. Meanwhile, the sex of many fish species is determined by a different mechanism. Females of these species have not the same, but different sex chromosomes (W and Z), and males, on the contrary, are the same (ZZ). Female heterogameticity of WZ leads to the fact that diploid gynogenetic offspring consists of fish with different sexual genotypes: WZ - females, ZZ - males and WW. The sex of fish with the WW genotype is still reliably unknown. The heterogeneity of the sexual composition of gynogenetic offspring does not allow using it to obtain homosexual offspring in a known manner.

The American paddlefish, with female homogameticity, belongs to the Polyodontidae family, and all other sturgeons are producers of black caviar (beluga, stellate sturgeon, sturgeon) - to the Acipenseridae family. It was shown that one of the representatives of the Acipenseridae family, the white sturgeon (Acipenser transmontanus), has female heterogeneity (Van Eenennaam AL, Van Eenennaam JP, Medrano JF, Doroshov SI Evidence of female hetero-gametic genetic sex determination in white sturgeon // J. Hered 1999b. V.90. P.231-233). The mechanism for determining sex in other sturgeon species has not yet been studied.

The technical result of the invention consists in the creation of clonally breeding lines of female sturgeon fish, which makes it possible to obtain homosexual offspring in fish with any sex determination mechanism, including when the mechanism is unknown, and makes it possible to significantly reduce costs in the formation of broodstock.

The indicated technical result is achieved by the fact that in the method for producing homosexual offspring in sturgeon fish, including the induction of the gynogenetic development of embryos by fertilizing the eggs with genetically inactivated sperm, the peculiarity is that the induction of gynogenetic development is applied to the eggs of female hybrids of phylogenetically distant species capable of producing unreduced diploid eggs, genetically identical to the mother.

The phenomenon of egg reduction is known in many species (natural forms) of fish (Vasiliev V.P. Evolutionary fish karyology. M: Nauka, 1985, 301 pp.). All of these forms are of hybrid origin. Such forms reproduce by natural gynogenesis, i.e. their eggs contain a special cytoplasmic mechanism that arose during evolution, which excludes the male set of chromosomes from development. Diploidy of the eggs determines the normal development of gynogenetic individuals.

Egg diploidy in natural hybrids and their gynogenetic descendants is due to a special transformation of meiosis, which consists in the fact that even before the first meiotic division begins, additional DNA replication (endoreduplication) occurs in the chromosomes, as a result of which the number of chromosomes doubles. In this regard, in the first division of meiosis, conjugation and recombination occur not between homologous, but genetically identical sister chromosomes. In addition to diploidy, the genetic consequence of such a transformation of meiosis is that all mature eggs are isogenic and identical to the mother’s genotype. In this they differ from ordinary fish, meiotic recombination in which leads to the genetic diversity (variability) of the produced eggs. The phenomenon of egg reduction does not occur in all hybrids, but only in the case of a certain (not too large and not too small) phylogenetic remoteness of the species that make up the hybrid combination.

The isogenicity of the eggs, their identity to the maternal genotype and the presence of a mechanism for inactivation of the sperm chromosome set (natural gynogenesis) leads to the fact that all individuals developing from such eggs are a kind of clone reproducing the mother’s genotype, i.e. clonal inheritance takes place. Therefore, this clone consists only of females and is, therefore, same-sex. The sexual composition of the offspring in this case does not depend on female homo- or hetero-gamety.

Preliminary studies have found that some artificial hybrids of phylogenetically distant sturgeon species are also capable of a similar transformation of meiosis, in which hybrid females produce more or less unreduced diploid eggs that are genetically identical to mothers. Therefore, in the inventive method for producing same-sex offspring in sturgeon fish, the induction of gynogenetic development is applied to the eggs of female hybrids of phylogenetically distant species. When these eggs are inseminated with genetically inactivated (irradiated) sperm, the resulting offspring is identical to the mother clone, and therefore all fish in the offspring are females.

Thus, the inventive method allows to obtain clonally propagating lines of female sturgeon fish, regardless of the sex determination mechanism. This makes it possible to form the broodstock, consisting of only females, with the aim of further mass production of sturgeon caviar. At the same time, there is no need to identify the sex, apply effects such as temperature shock, hormonal processing to the embryos and fish, and the costs of raising males are reduced. Obtaining clonal breeding lines of females also has the advantage that, when cloning the first generation of hybrids, all subsequent generations are genetically identical to the first, preserving its fishery indicators, while usually sturgeon hybrids are most productive in the first generation, and their productivity decreases in subsequent generations. In addition, when using the proposed method solves the problem of biosafety, because in the case of clonal hybrid females getting into natural water bodies and their crosses with close pure species, the offspring will be triploid and therefore sterile.

Thus, the combination of distinctive features ensures the achievement of the specified technical result.

As a result of the analysis of the prior art, no analogues are found that are characterized by features identical to all the essential features of the claimed invention. Therefore, the claimed invention meets the condition of "novelty."

The new set of characters ensures the achievement of a new technical result and does not follow explicitly from the prior art for a specialist, since never before the same time, sturgeon fish and vertebrate animals in general have artificially obtained same sex offspring by cloning females with an unknown sex determination mechanism. Thus, the claimed invention meets the condition of "inventive step".

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

The method is as follows.

Hybrids of sturgeon fishes of phylogenetically distant species are obtained, the females of which are capable of producing diploid unreduced eggs. At the same time, hybrid females are chosen whose eggs are larger than usual (diploid eggs are larger than haploids), or consist of a mixture of large (diploid) and small (haploid) eggs. The sperm of one of the parent species or the male hybrid is irradiated with ionizing or UV radiation in a dose that causes complete genetic inactivation of all sperm. The eggs of hybrids are seeded with irradiated sperm. In this case, the induction of the gynogenetic development of the embryos occurs. The use of temperature shock for the diploidization of gynogenetic haploid embryos is not required, since the eggs in which the premeiotic chromosome endoreduplication has occurred are already diploid. The vast majority of gynogenetic embryos developing from reduced eggs will remain haploids and die. Thus, by the yield of viable gynogenetic larvae, one can judge the frequency of endoreduplication in this hybrid combination and in this hybrid female.

Diploid viable gynogenetic larvae can arise in two ways: due to spontaneous suppression of the second division of meiosis or due to diploidy of the oocytes due to DNA endoreduplication. It is proved that the frequency of larvae appearance due to spontaneous suppression of the second division of meiosis in sturgeon fishes is very small and amounts to about 0.1% (Rekubratsky A.V., Grunina A.S., Barmintsev V.A., Golovanova T.S., Chudinov O .S., Abramova AB, Panchenko NS, Kupchenko SA Meiotic gynogenesis in stellate sturgeon, Russian sturgeon and sterlet // Ontogenesis. 2003. V.32. No. 2. S.121-131). These larvae will not be isogenic and may differ from the female in individual genetic characteristics. They can develop in both females and males. The larvae that arose due to the diploidy of the eggs due to DNA endoreduplication are a clone genetically identical to the mother. Of these, only females can develop.

Growing and gynogenetic reproduction of hybrid females, capable of producing diploid eggs with a high frequency, allows the formation of a broodstock, from which it will be possible to obtain homosexual sturgeon offspring on an industrial scale.

An example implementation of the method.

The method is implemented on hybrids between beluga and sterlet (hybrid bester). Besters have been received for quite some time and are used in aquaculture. The choice of this hybrid of sturgeon fishes was made on the basis of data on the fertility, fecundity, and size of the eggs of the bester, which suggested that some of the eggs produced by the female bester are diploid, i.e. their chromosome set was formed as a result of premeiotic chromosome endoreduplication.

From 15 female bester, 5 female sterlet and 2 female beluga got caviar. Sperm was obtained from 5 male bester and mixed it. Equal portions were taken from the eggs obtained from each female, which were used in the experiment for setting crosses. After receiving gametes from all males and females, fin fragments were taken and fixed in 96% ethanol for subsequent molecular genetic analysis. Part of the sperm was trained with shortwave UV light for 7 minutes. Preliminary studies have established that such a procedure causes complete genetic inactivation of sperm, however, after irradiation, sperm retain mobility and are able to activate the eggs to gynogenetic development.

Using the eggs of each of the females, the following crosses were made:

(1) female bester × male bester, insemination with normal sperm (control);

(2) female bester × male bester, insemination by irradiated sperm;

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

(4) female sterlet × male bester, insemination by irradiated sperm;

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

(6) female Beluga × male Bester, insemination with normal sperm (control);

(7) female Beluga × male Bester, insemination by irradiated sperm.

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

Embryos were incubated in Petri dishes at a temperature of 20 ° C. 5-10 min after insemination of the embryos from crosses 5 and 8 was subjected to heat shock at a temperature of 37 ° C for a duration of 2.5 minutes. Heat shock at this point in development inhibits the second division of meiosis in embryos, thereby ensuring the restoration of diploidy. Incubation lasted 7 days. Hatching of larvae lasted 2 days. The transition of larvae to active nutrition occurred 6–9 days after hatching.

The following results were obtained in crosses.

In all crosses of the control variants (1, 3, 6), normal prelarvae were obtained (yield from the number of fertilized eggs from 65 to 85%), most of which (75-90%) continued to develop normally and started active feeding.

Almost all embryos from crosses in which sterlet and beluga caviar were seeded with irradiated sperm (variants 4 and 7) developed abnormally, they had clear signs of haploid syndrome (axial skeleton curvature, pericardial hydration, threadlike heart). All embryos from crossing 7 died before hatching. Most of the embryos from option 4 also died before hatching. In total, among the hatched larvae (0–2% in different crosses), only 4 morphologically normal specimens were found (0.005%). All ugly prelarvae died within a few days after hatching, even before switching to active nutrition. Two out of four normal larvae started active feeding. They could arise due to the spontaneous suppression of the second division of meiosis in fertilized eggs, which led to the restoration of the diploid status of gynogenetic embryos.

In variants 5 and 8, in which heat shock was used to diploidize the gynogenetic haploids of sterlet and beluga by suppressing the second division of meiosis, normal diploid larvae were obtained (10-15%).

In the crosses of option 2, in which irradiated sperm inseminated the eggs of the bester, a large number of normally developing embryos were noted. The yield of morphologically normal prelarvae in different crosses (eggs of different females) ranged from 3 to 25%. Most of them (65-85%) started active nutrition.

Experienced (173 pcs., Option 2) and control (50 pcs., Option 1) bisters were grown in aquariums. In addition, 30 pieces were grown. diploid gynogenetic individuals of sterlet (option 5) and beluga (option 8). At the age of 2 months, fin fragments were taken from all fish for intravital molecular genetic analysis. DNA was extracted from the fins of juveniles and their parents by the standard method. The RAPD spectra of total DNA and the polymorphism of microsatellite sequences (4 loci) were investigated.

The analysis showed that the DNA of the control fish (option 1) contained alleles of microsatellite loci that were present in both mothers and fathers, and their RAPD spectra were a combination of bands characteristic of both parents. Thus, in this embodiment, a pattern was observed that occurs with ordinary crossbreeding.

The frequency of homozygotes at microsatellite loci among gynogenetic individuals of sterlet and beluga (variants 5 and 8) was significantly higher than in the control. Gynogenetic individuals differed from each other and also differed from mothers both in sets of microsatellite alleles and in RAPD spectra. Genetic differences in the gynogenetic progeny of pure species are caused by divergence of homologous chromosomes into different germ cells and by recombination of chromosomes during meiosis.

In the experimental version (2), the majority of individuals (80-90%) are identical in terms of the studied parameters and do not differ from their mothers, i.e. they can be identified as maternal clones. This situation could arise only if part of the eggs of the female bester was diploid due to DNA endoreduplication in early meiosis. All such fish can only be females. Identified genetic differences in a small part of the experimental fish can be caused by changes in DNA endoreduplication, as well as mutations. Bester females, which had the highest yield of gynogenetic diploid prelarvae, were selected for further reproduction with the aim of forming a broodstock, from which it would be possible to obtain homosexual sturgeon offspring on an industrial scale.

Thus, the claimed method allows to obtain clonally propagating lines of female sturgeon fish (same sex offspring) regardless of the sex determination mechanism and thereby reduce material costs in the formation of broodstock. In a similar way, same-sex offspring can be obtained not only from the bester, but also from hybrids between other species of sturgeon, provided that these hybrids are capable of producing unreduced gametes genetically identical to the maternal genotype.

The above information indicates that the method for producing homosexual sturgeon offspring according to the claimed invention relates to genomic engineering in the field of fish farming and, in particular, is aimed at solving the problem of sex regulation in sturgeons. For the claimed method in the form as described in the claims, the possibility of its implementation using the means and methods described in the application is confirmed. Therefore, the claimed invention meets the condition of "industrial applicability".

Claims (1)

A method for producing homosexual offspring in sturgeon fish, including the induction of the gynogenetic development of embryos by fertilizing eggs with genetically inactivated sperm, characterized in that the induction of gynogenetic development is applied to the eggs of female hybrids of phylogenetically distant species capable of producing unreduced genetically diploid egg cells.