CN111172189B - Establishment method of Sissy medaka model for simulating human sperm ovarian syndrome - Google Patents

Abstract

The invention relates to a method for establishing a Sissy medaka model for simulating human spermatic ovarian syndrome, which comprises the following steps: a) Breeding to obtain the gsdf KO medaka of the homozygous female fish with the gsdf knocked out by using the gsdf specific primer; b) Utilizing crystalline lens co-expression gsdf2AEGFP plasmid to establish gsdf ectopic expression transgene XX male species fish Tg, cryGsdf2A-EGFP; c) Genetic hybridization and breeding: the method comprises the steps of obtaining a gsdf heterozygous XXTg male medaka through the female hybridization of a cryGsf 2A-EGFP transgenic XX male fish and a gsdfKO, and finally obtaining the hermaphrodite Sissy medaka through the hybridization of the gsdf heterozygous XXTg male medaka and the gsdfKO female medaka. The invention establishes an animal model of the spermatid ovary by utilizing gene editor for the first time, and provides a living animal model for researching a molecular mechanism of female and male differentiation of germ cells and a pathogenesis of spermatid ovary generation.

Description

Establishment method of Sissy medaka model for simulating human sperm ovarian syndrome

Technical Field

The invention relates to establishment of a disease model experimental animal model, in particular to an establishment method of a Sissy medaka model for simulating human sperm ovarian syndrome.

Background

The gonadal development of human beings and animals is abnormal due to genetic or epigenetic factors, environmental society and other factors, so that fertility or reproduction rate is obviously lower than that of normal individuals, gonadal cells, gonadal structure and function abnormality, tumor generation and other pathological changes, and the reproductive health of human beings and animals is increasingly influenced and threatened in recent years. Common but unknown pathogenesis is that human sperm ovarian signs, gonadal dysplasia or testis cancer is due to the fact that the male sex of humans is determined by the SRY male determinants of the Y chromosome, but there are still many undiagnosed puzzles on how SRY coordinates other factors, together with the regulatory mechanism that regulates the final entry of germ cells into male differentiation and development.

The development of male sterility treating method and contraceptive medicine needs corresponding animal model to develop deeply.

Patent document CN103361336a, publication date 2013.10.23, discloses a male fertility disorder animal model of a non-human mammal, a preparation method and use of the animal model, and the functions of the Prss37 gene and protein are identified. The Prss37 gene of the animal model is inactivated, thereby affecting the appearance of ADAM3 in mature sperm produced by the model animal, indirectly affecting sperm-zona pellucida binding and sperm migration from uterus to oviduct, resulting in serious male fertility disorders. The model and Prss37 gene and proteins thereof can be used for screening ADAM3 maturation promoters and infertility treatment agents or contraceptives.

However, to date, animal models suitable for studying the developmental association of spermatogenesis are extremely rare.

The inventor of the application published a review paper in period 6 of journal heredity 2017 "overview of sex determination and sexual differentiation research progress of teleost fish with model organism", and disclosed that "the laboratory utilizes gene knockout or transgene over-expression gsdf, a growth transforming factor (TGF-beta), to realize regulation and control of male and female differentiation of medaka. We have also found that gsdf is not only a key factor in initiating the male differentiation pathway, but also acts on the early development of egg cells and regulates the expression of other related genes in the pituitary-gonadal axis reproductive endocrine feedback system ", but how to construct a refined ovarian hermaphrodite animal model remains a challenge.

Disclosure of Invention

The invention aims at overcoming the defects in the prior art, and provides a method for establishing a Sissay medaka model for simulating human spermatic ovarian syndrome.

It is a further object of the present invention to provide the use of the sisty medaka model.

In order to achieve the first object, the invention adopts the following technical scheme:

a method for building a Sissy medaka model for simulating human spermatic ovarian syndrome, comprising the following steps:

a) Breeding to obtain the gsdf knockout homozygote female fish gsdfKO female medaka by using the gsdf specific primer;

b) Separating XX transgenic male breeding fish with gsdf ectopic expression by using green fluorescence of crystalline lens: creating and linearizing a p817-gsdf2A-EGFP expression plasmid, and microinjecting fertilized eggs to obtain a stable inherited Tg: cryGsdf2A-EGFP transgenic line;

c) Genetic hybridization protocol: obtaining a gsdf heterozygous XXTg male medaka through the female hybridization of the cryGsf 2A-EGFP transgenic XX male fish and the gsdfKO, and finally obtaining the gsdfKO transgenic medaka, namely the Sissy medaka model through the hybridization of the gsdf heterozygous XXTg male medaka and the gsdfKO female medaka.

The sequence of the p817-gsdf2A-EGFP expression plasmid is shown in SEQ ID NO. 3.

The gsdfKO female medaka is obtained by knocking out endogenous gsdf with a zinc finger nuclease specific target.

The fertilized egg is a fertilized egg of 1 cell stage.

In order to achieve the second purpose, the invention adopts the following technical scheme:

the use of the Sissy medaka model selected from the group consisting of:

a) Research on molecular mechanisms related to sex determination and differentiation of human or animal;

b) Screening suspected intrinsic or extrinsic environmental factors affecting the control of developmental differentiation of human or animal germ cells.

The animals cover lower to higher vertebrates including fish, amphibians, reptiles, birds, and mammals including mice, dogs, rabbits, monkeys, and humans.

The invention has the advantages that:

according to the invention, an animal model of the hermaphrodite sperm ovary, namely a Sissy medaka model, is successfully established for the first time, in the model, gsdf is introduced into local cells of homozygotes with the gsdf removed from genome, so that uneven distribution of gsdf signal expression cells is caused, disorder of microenvironment of germ cells and surrounding somatic cells is influenced, and germ cells under the action of the somatic cells with positive gsdf expression are promoted to enter a spermatogenic pathway; germ cells under the action of gsdf expressing negative somatic cells enter the ovum production process, eventually leading to the formation of both sperm and immature ovum cells in the same gonad.

1. The invention lays a foundation for researching the molecular mechanism research of a signal regulation path in the sex conversion process of the natural hermaphrodite fish in female-male differentiation development of the germ cells of fish, expects to reveal how different fish sex determination mechanisms of hermaphrodite and/or hermaphrodite lead the germ cells to finally enter the mutual association of hermaphrodite differentiation to evolve a conserved molecular mechanism, establishes a model of how related genes and proteins interact to form molecular network regulation, and provides theoretical basis for sex control and genetic operation of the fish. The invention is also convenient for screening chemical or/and environmental endocrine disruptors affecting the differentiation of male and female gonads of fishes in river water bodies.

2. In vertebrate phylogenetic evolution, fish are in a critical position after the front-opening. Compared with higher vertebrates, the sex determination mechanism of fish has originality, diversity and variability, and has sex determination modes of all vertebrates, various sex types from hermaphrodite to hermaphrodite exist, sex reversal is a more common phenomenon in fish, and therefore, research on the sex determination mechanism of fish has very important theoretical value for the formation of the sex determination mechanism of the whole vertebrate group and the disclosure of evolutionary pathways. The Sissy refined ovarian hermaphroditic medaka provided by the invention provides a living platform and a test material for further screening related suspected internal or external environmental factors influencing and controlling development and differentiation of germ cells, analyzing pathogenic mechanisms of pathological changes such as fertility decline or abnormal proliferation, and the like, is beneficial to analyzing the pathogenic mechanisms and mechanisms of infertility caused by overall low fertility trend and increasing infertility of people and animals, and discussing solutions and countermeasures.

Drawings

Fig. 1: and (5) genotyping.

Fig. 2: establishment of expression vectors and transgenic medaka lines for p817-gsdf 2A-EGFP. Gsdf is overexpressed in the lens of transgenic medaka. Schematic representation of the expression vector of (a). EGFP is expressed in the embryonic lens from 4 days post fertilization to adulthood. Eighth day after fertilization, embryos were examined at low power (b) and high power (b'); f0 generation (c and d) 3 months old transgenic fish.

Fig. 3: expression of EGFP signal in HPG axis.

Fig. 4: co-expression of the gsdf and cyp19b genes in WT XY male and Tg: cryGsdf2A-EGFP XX male brains. ICH analysis was performed with anti-Cyp 19b and Gsdf antibodies for WTYX (b is a magnified image of b') and Tg: cryGsdf2A-EGFP XX. Endogenous Gsdf was found mainly in the supraoptic nucleus (SC) and a small number of Cyp19b positive neurohypophysis cells (arrows). In both the pituitary and hypothalamus of transgenic individuals, both cyp19b positive and negative cells were able to observe an anti-Gsdf response signal (arrow), demonstrating that Gsdf is expressed in these cells.

Fig. 5: two-step genetic scheme. Obtaining XX male from the Tg cryGsdf2A-EGFP transgenic line, and obtaining gsdf mutant XY female by gsdf knockout; the gsdf homozygote and heterozygote transgenic individual are hybridized to finally obtain Sissy (gsdf-/-Tg: cryGsdf2A-EGFP XY).

Fig. 6: gsdf-/-XY male and normal XY female.

Fig. 7: silsy hermaphrodite appearance.

Fig. 8: testis of transgenic XX male.

Fig. 9: sissy refined ovaries. The green fluorescence (EGFP) signal can be detected by the lens of the transgenic individual; ruler = 1mm.

Fig. 10: histological analysis of cryGsdf2A-EGFP XX testis and Sissy ovary. (a) Tg cryGsdf2A-EGFP XX spermatic. (a') is an enlarged image of (a). (b) Spermatogenesis and oogenesis coexist in one spermatic ovary. And (b') is (b) a magnified image. Sg I, spermatogonial cell type I; sg II, spermatogonial cell type II; sc, spermatocyte; st, sperm cells; sz, sperm; od, double-line oocyte; op, pachytic oocytes; GC, germ stem cells (arrow).

Fig. 11: the expression of gonadal androgens (estradiol E2, testosterone T) synthesizing key enzyme (17 alpha-hydroxylase cyp17-a1; ovarian aromatase cyp19 a) and the level of androgens in blood. (a) cyp19a and cyp17a1 are key enzymes for E2 and T production and conversion, and are highly expressed in the ovary or testis, respectively. (b) The E2/T ratio in the serum of the male is lower and the E2/T ratio in the serum of the female is higher. All data were taken from serum mixtures of n=6 to 10 independent individuals and the ±se (n=3) was determined in more than three experimental replicates. * p < 0.01.WT: wild-type normal gsdf +/+; hm: knockout homozygote gsdf-/-; sissy: gsdf-/-Tg.

Fig. 12: transgenic overexpression of Gsdf signals stimulated the expression of follicle stimulating hormone (Fsh) receptor and brain aromatase (cyp 19 b).

Detailed Description

The following detailed description of the invention provides specific embodiments with reference to the accompanying drawings.

Example 1

1 Sissy medaka model establishment

1.1 breeding to obtain the homozygous female parent fish with the gsdf knocked out by using the gsdf specific primer

The medaka is fed in a water circulation system at 26-28deg.C, and the light and dark circulation time is 14/10 hr, and the treatment is strictly performed according to the instruction of the experimental animal research Commission of Shanghai university. Endogenous gsdf was knocked out with zinc finger protein nucleases (zinc-finger nucleic acids) specific targets, see previous report [1]. Assessment of sex phenotype was confirmed by secondary sex characteristics (dorsal and gluteal fin shapes) and gonadal anatomy. The primers (PG 17.55'-CCGGGTGCCCAAGTGCTCCCGCTG (SEQ ID NO: 4); PG 17.6.5' -GATCGTCCCTCCACAGAGAAGAGA (SEQ ID NO: 5)) were able to specifically amplify dmy and autosomal dmrt1 of the medaka Y chromosome, for the identification of XX and XY genotypes [2]. gsdf genotyping was performed according to our previously published paper [1].

Results: PCR amplification of genomic DNA was performed using a pair of primers (PG 17.5-17.6) to give XX pattern containing only 1.1kb band from dmrt1 and XY pattern containing both bands of dmrt1 and dmy (0.9 kb). Identification of gsdf wild type, heterozygotes and homozygotes, increased by 4 nucleotide bases based on the knockout mutant, forms a heterozygous band with the wild type allele, and homozygote DNA, when wild type DNA was not added, did not produce a heterozygous band, but after wild type DNA was added, a heterozygous band appeared to distinguish the genotype of the wild type individual (FIG. 1).

1.2 sorting out XX transgenic Male species of Fish with ectopic expression of gsdf by Green fluorescence of lens

Because gonads, pituitary glands and eyeballs are developmentally originated from the same cell line [3], microinjection of the mouse lens protein (gamma-crystallin) promoter drives the expression vector of green fluorescent protein (EGFP), which can produce transgenic medaka [4] with the lens expressed as green fluorescent EGFP. To investigate how gsdf modulates the molecular mechanism of gonadal differentiation via the hypothalamic-pituitary-gonadal axis (HPG), we performed the following redesign with the existing p817-EGFP expression plasmid. EGFP in p817-EGFP ([ 4] Addgene) is replaced by Gsdf2A-EGFP fragment, and Gsdf2A-EGFP fusion protein is introduced by using a 2A peptide-mediated co-expression system, so that green fluorescence-expressed cells can simultaneously overexpress Gsdf. The expression vector is injected by micro, so that the transgenic line Tg cryGsdf2A-EGFP is established. The resulting transgenic individuals showed green fluorescence of the lens from embryonic to adulthood, with continuous expression of Gsdf and EGFP on the HPG axis (fig. 2). The specific construction method of the vector is as follows:

the vector skeleton is taken from Dr Kozmik, constructed and published on an Addgene webpage, and p 817-mgama Fcry-EGFP (4280bp,ID 23156,www.addgene.org), called p817-EGFP for short. We linearized the vector p817-EGFP with EcoRI and NcoI endonucleases using PCR and In-Fusion HD Cloning

(Clontech), the Gsdf-2A fragment was inserted in front of EGFP to give p817-Gsdf2A-EGFP, the full length of which was 4971 bp. The sequence of the synthetic vector segment adhesion primer is as follows:

gsdfFw1：5’-cttcccatcgattcgatgtctttggcactc(SEQ ID NO:1)

2ARv2：5’-GCCCTTGCTCACCATGGGCCCAGGGTTTTC(SEQ ID NO:2)

the gsdf2A fragment was amplified by PCR and ligated to the p 817-mgama Fcry-EGFP vector, named p817-gsdf2A-EGFP, with the pattern shown in FIG. 2A and the complete sequence shown in SEQ ID NO: 3. Sequencing confirms that the constructed expression plasmid is accurate.

By Invitrogen ^TM Plasmid DNA was extracted and the p817-gsdf2A-EGFP expression plasmid was linearized with I-SceI DNA endonuclease (Takara Dailian, china). Microinjection into the 1-cell stage of fertilized eggs was performed at a concentration of 50 ng/. Mu.l of DNA. Foundar species fish capable of being integrated and reproductive transferred to the offspring genome are obtained, transgenic individuals with green fluorescence at the lens at the beginning of embryo Stage (Stage 33) are obtained through genetic hybridization and PCR reaction can also detect fragments with corresponding sizes, and the gsdf2A-EGFP is proved to be integrated into the genome of the individuals. The mouse monoclonal antibodies against Gsdf and GFP detected positive expression cells in both brain and gonads, suggesting that these cells expressed both transgenic Gsdf and EGFP proteins.

Results: from the 1256 fertilized eggs injected, 17 sexually mature medaka were obtained, the lenses of which exhibited GFP fluorescence signals from the beginning of the embryo period (Stage 33) (b and b' in FIG. 2), and these transgenic fluorescence signals were continuously expressed to adults (c and d in FIG. 2).

The overall viability and transgene efficiency of the injected embryos are summarized in table 1. After 5 of them were selected to be outcrossed with wild females and males, 3F 0 Foundator fish (1 male fish and 2 female fish) transmitted GFP signals to offspring, indicating that transgenic expression of pGsdf2A-EGFP did not affect gonadal development. We note that GFP signals appear to be less sexual in F0 fish than in females (c and d in FIG. 2). No phenomenon similar to this sex difference was reported in the previous p817-EGFP Cab and Heino lines [4]. Mouse γF-lens driving factors regulate expression of lens proteins by local cell lines other than the lens, including retina, brain and testis [3]; we also detected gsdf-GFP signal in the brain and gonads (ovaries or spermary) of transgenic individuals (fig. 3), whereas no fluorescent signal was detected in the wild-type female and male brains or gonads as controls (fig. 3).

TABLE 1 Total survival and transgenic efficiency of p817-gsdf2A-EGFP medaka

¹⁾ A linear or circular expression plasmid DNA (pmγcry-gsdf2AEGFPDNA,40 ng/. Mu.l) was microinjected.

²⁾ The percent survival was determined using the number of fertilized eggs injected as the denominator.

³⁾ And (3) performing outcrossing on fish with green fluorescence on eyeballs and normal wild medaka, and detecting fertility of the fish.

⁴⁾ Can transmit green fluorescence transgene signals to offspring's breeding fish.

Three Tg's, cryGsdf2A-EGFP transgenic families can obtain 17.4-44.4% XX males with different proportions in four filial generations of wild type, which indicates that the overexpression of gsdf in brain or/and gonad can promote XX male differentiation. In the F1 generation, 7/32 (21.9%) and 12/28 (42.9%) XX males were from two independent transgenic lines (Table 2). These transgenic F1 males were further hybridized with WT females, yielding approximately 50% gfp-positive XX males. Not all XX Tg, cryGsdf2A-EGFP, becomes XX male, presumably the local gsdf expression does not exceed the threshold required for XX maleation, or gsdf must be expressed in specific cells during critical stages of sexual differentiation, and the male pathway is initiated to promote XX maleation. In the Tg cryGsdf2A-EGFP line, most of hypothalamus and ovaries were GFP positive and pituitary GFP negative were XX female, whereas over 78% of XX males were hypothalamus-pituitary-testis (HPT) GFP positive (c 1-c4 in FIG. 3), consistent with our guessed local specific cell expression gsdf, which resulted in XX maleation hypothesis. Endogenous gsdf was detected in WTXX and XX Tg: cryGsdf2A-EGFP male medaka in the suprachiasmatic nucleus (SC) as shown in fig. 4 a and b, weakly in a few cells coexpressed with cyp19b in the pituitary region (arrow in fig. 4), or only gsdf positive cells in XX Tg: cryGsdf2A-EGFP male medaka (arrow in fig. 4), but not in WT females (data not shown).

TABLE 2 XX Male development of transgenic medaka

ND means no further tracking.

1.3 genetic hybridization protocol

The Sissy medaka is derived from the HdrR strain. XYgsdf KO female medaka was first obtained by hybridization between gsdf heterozygotes. Stable inherited Tg cryGsdf2A-EGFP transgenic lines were obtained by microinjection of fertilized eggs. And then obtaining the gsdf heterozygous XXTg male medaka by hybridizing the XX male fish with the Tg cryGsdf2A-EGFP transgene with the gsdfKO female. And hybridizing with the female medaka of the gsdfKO to finally obtain the gsdfKO transgenic medaka, i.e. Sissy.

Results: we used a two-step genetic hybridization protocol to generate Tg: cryGsdf2A-EGFP gsdf from initial mating of a male fish of Tg: cryGsdf2A-EGFPXX with a homozygous XY female of gsdf and continuous breeding of XX Tg: cryGsdf2A-EGFP male ^+/- Male and XY gsdf ^-/- Female (fig. 5). A Tg of cryGsdf2A-EGFPgsdf is obtained ^-/- XY medaka and named Sissy. genotyping of gsdf and dmy is summarized in table 3. 11 Tg's from 61 offspring cryGsdf2A-EGFP XYgsdf ^-/- Male and 8 Tg cryGsdf2A-EGFPXXgsdf ^-/- Female, following mendelian genetic law 1:1 (heterozygote: homozygote=31:30) separation (table 3). These 11 Sissy groups all exhibited male secondary sex characteristics, with their hip fins in the shape of parallelograms (FIGS. 6-7), all possessing a large abdomen. Dissections showed large abdominal cavities because their gonads were 4-5 times larger than normal XY or XX male testis (fig. 8-9).

TABLE 3 Tggsdf ^+/- XY and gsdf ^-/- Offspring genotyping of XX (male)

Gonadogenesis and spermatogenesis of medaka 2Sissy and sperm development are combined in the sperm ovary (Tg: cryGsdf 2A-EGFPgsdf) ^-/- )

Histological: the gonads of Sissy medaka and normal control group were fixed with Bouin's fixative (Picric acid: formaldehyde: glacial acetic acid 15:5:1), frozen, paraffin or resin embedded, and subjected to conventional or ultrathin sections. The thickness of a conventional tissue section is 5 μm, and the thickness of an ultrathin section observed by a transmission electron microscope is 60nm. Hematoxylin Eosin (HE) staining routinely observes cell morphology. Polychromatic fluorescence immunohistochemical analysis, observation and imaging analysis were performed with confocal laser scanning microscopy (Leica DMi8 TCS SP8, germany).

Results: histological analysis showed that the distal peripheral layer of the testis was thicker than the normal testis (a and a' in fig. 10), and that the spermatogonia were significantly proliferated. Although germ cells at different stages of development are packed during the outside-in seminiferous process from distal peripheral spermatogenic cells to proximal end spermatogenesis (b and b 'in fig. 10), there are also a large number of primary oocytes interspersed between seminal vesicles (b' in fig. 10), exhibiting the seminal ovarian phenomenon of concurrent egg production and spermatogenesis in the same gonad. In all 11 Sissy individuals we dissect, refined ovarian signs were present. Only primary oocytes that did not enter vitelline formation were not seen for secondary oocytes that entered vitelline formation, indicating that vitelline formation and/or vitelline absorption was regulated by Gsdf signals. We evaluated fertilization rates and embryo viability for Sissy sperm and the results are summarized in Table 4. In comparison with normal XY male testis, hermaphrodite Sissy was identical to normal female (wild type gsdf ^+/+ Or heterozygotes gsdf ^+/- ) The fertility of the fertilized eggs and the proportion of surviving embryos obtained by hybridization are significantly lower. The fertility and fertilized egg survival rate of Sissy sperm decreased to 25.58% -45.3% by 100% of fertilized eggs produced by normal group XX females and XY males of intact gsdf were not identical to those of the previously reported gsdf, which resulted in reduced fertility and embryo dysplasia, and the result of decreased survival rate was consistent [5]]。

TABLE 4 fertilization rates of Sissy medaka Male and Normal XY Male vs embryo survival rates

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The absence of 3gsdf signal affects the estradiol/testosterone (E2/T) hormone level in blood and the ratio of the two

Real-time quantitative RT-PCR detection shows that 17-hydroxylase (cyp 17a 1) necessary for gonadal estrogen and androgen synthesis is highly expressed in male testis and positively correlated with testis development; while the expression level of the ovarian aromatase cyp19a, a key rate-limiting enzyme for estrogen synthesis, is closely related to female egg cell development (A in FIG. 11).

The loss of gsdf directly disrupts the differentiation and development of granulosa cells surrounding the egg cells, reducing the number of granulosa cells, thinning the granulosa cell layer, leading to a decrease in the expression of cyp19a produced by granulosa cells, leading to a barrier in oocyte development [5]. The expression of cyp19a was significantly lower than female levels, approaching male, in both the testis and the Sissy sperm ovaries of the transgene Tg cryGsdf2A-EGFP XX (FIG. 11A). It has been reported that the deletion of cyp19a, while not directly affecting early egg cell production, is essential for maintaining egg cell development and maturation [6]. Post-maleation of XX gonads after Cyp19a knockout occurs, producing XX testis, indicating that a decrease or deletion of Cyp19a eventually leads to maleation of germ cells and somatic cells [6].

The levels of E2 and T in serum were determined using an enzyme immunoassay kit and the results are summarized in FIG. 11B as described previously. Serum E2 and T levels were close to those of normal XY males (B in FIG. 11) in comparison to normal females and males, tg cryGsdf2A-EGFP XX males, consistent with the male phenotype of the second sex. Sissy individuals E2 and T are between normal females and males, and also conform to the male-female phenotype of the second sex characteristic hip fin, while the dorsal fin is female. The E2 to T ratio in normal female blood is almost 100 times that in normal male (B in FIG. 11), while the E2/T ratio in gsdf knocked-out females or Sissy is between normal male and female. In particular, the T levels of either XY or XX females with a loss of Gsdf were not significantly different from normal XX females, whereas XX males produced by overexpression of the Gsdf transgene, and blood T levels of the Sissy hermaphrodites were significantly higher than females, reflecting that the Gsdf signal could directly or indirectly raise testosterone levels (B in FIG. 11) consistent with male external characteristics (hip fin shape) (FIGS. 6-8).

qPCR also aligned amh/amhrII and fsh/lh receptor expression profiles from the normal and gsdf deleted groups. The transgenic gsdf signal was found to promote expression of cyp19b in the gonads (a in fig. 12). The levels of amh and amhrII expression were higher in the normal testis than in the ovary, were inhibited in the Gsdf knockout ovary, but were high in the Tg: cryGsdf2A-EGFP testis, indicating that amh and amhrII expression were also regulated and affected by the Gsdf signal (FIG. 12A). The Gsdf signal also inhibited the expression of follicle stimulating hormone receptor (fshr) and luteinizing hormone receptor (lhr) in the gonads (B in fig. 12). We found that lhr increased significantly in both Sissy and Tg: cryGsdf2A-EGFP XX males, whereas fshr decreased significantly in Gsdf-deleted ovaries, and increased significantly in Sissy, indicating that fshr expressing cells responded more strongly to Gsdf signals relative to lhr expressing cells (B in fig. 12).

Reference is made to:

1.Zhang,X.,et al.,Autosomal gsdfacts as a male sex initiator in thefish medaka.Scientific Reports,2016.6.19738.

2.Matsuda,M.,et al.,DMYis a Y-specific DM-domain gene requiredfor male development in the medakafish.Nature,2002.417(6888):p.559-63.

3.Magabo,K.S.,et al.,Expression ofbetaB(2)-crystallin mRNA andprotein in retina,brain,and testis.Invest OphthalmolVis Sci,2000.41(10):p.3056-60.

4.Vopalensky,P.,et al.,A lens-specific co-injection marker for medaka transgenesis.Biotechniques,2010.48(3):p.235-6.

5.Guan,G.,Sun K.,Zhang X.,Zhao X.,Li M.,Yan Y.,Wang Y.,Chen J.,Yi M.,and Hong Y.,Developmental tracing of oocyte development in gonadal soma-derived factor deficiency medaka(Oryzias latipes)using a transgenic approach.Mechanisms ofDevelopment 2017.143:p.53-61.

6.Nakamoto,M.,et al.,Ovarian aromatase loss-of-function mutant medaka undergo ovary degeneration andpartialfemale-to-male sex reversal afterpuberty.Mol Cell Endocrinol,2018.460:p.104-122.

the foregoing is merely a preferred embodiment of the present invention, and it should be noted that modifications and additions may be made to those skilled in the art without departing from the method of the present invention, which modifications and additions are also to be considered as within the scope of the present invention.

SEQUENCE LISTING

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gggatcactc tcggcatgga cgagctgtac aagtaaagcg gccgcgactc tagaactata 1920

gtgagtcgta ttacgtagat ccagacatga taagatacat tgatgagttt ggacaaacca 1980

caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat 2040

ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt 2100

ttcaggttca gggggaggtg tgatatcaag cttatcgata ccgtcgacct cgaggggggg 2160

cccggtaccc aattcgccct atagtgagtc gtattacgcg cgcattaccc tgttatccct 2220

atcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa 2280

tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga 2340

tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc 2400

attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct 2460

agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg 2520

tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga 2580

ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt 2640

ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg 2700

aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc 2760

ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat 2820

attaacgctt acaatttagt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt 2880

tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc 2940

ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc 3000

ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa 3060

aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg 3120

gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag 3180

ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc 3240

gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta 3300

cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg 3360

cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca 3420

acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac 3480

caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat 3540

taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg 3600

ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata 3660

aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta 3720

agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa 3780

atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag 3840

tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg 3900

tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact 3960

gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg 4020

taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc 4080

aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata 4140

ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta 4200

catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc 4260

ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg 4320

ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac 4380

agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg 4440

taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt 4500

atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct 4560

cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg 4620

ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata 4680

accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca 4740

gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc 4800

gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg 4860

agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta 4920

tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac a 4971

<210> 4

<211> 24

<212> DNA

<213> artificial sequence

<400> 4

ccgggtgccc aagtgctccc gctg 24

<210> 5

<211> 24

<212> DNA

<213> artificial sequence

<400> 5

gatcgtccct ccacagagaa gaga 24

Claims (2)

1. A method for building a Sissy medaka model for simulating human sperm-ovarian syndrome, comprising the following steps:

a) Breeding of a homozygous female fish with a gsdfKO female medaka (XYgsdf) by using a gsdf-specific primer ^-/- ♀)；

b) Tg crigsdf 2A-EGFP transgenic XX male fish (XXgsdf) was sorted using lens green fluorescence ^+/+ And (3) the following steps: constructing and linearizing a p817-gsdf2A-EGFP expression plasmid, and microinjecting fertilized eggs to obtain a stable inherited Tg cryGsdf2A-EGFP transgenic line, wherein the sequence of the p817-gsdf2A-EGFP expression plasmid is shown as SEQ ID NO. 3, and the fertilized eggs are fertilized eggs in 1 cell stage;

c) Genetic hybridization protocol: XX Male fish (XXgsdf) transgenic for cryGsdf2A-EGFP by Tg ^+/+ Female) and gsdfKO female (XYgsdf) ^-/- (ii) crossing to obtain a gsdf heterozygous XXTg male medaka (XXgsdf) ^+/- Female) and then with gsdfKO (XYgsdf) ^-/- (ii) the final obtaining of gsdfKO transgenic medaka, sissy

A medaka model.

2. The method of claim 1, wherein the gsdfKO female medaka is obtained by knockout of endogenous gsdf with a zinc finger nuclease specific target.

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