CN107099501B - Method for inducing young primates to obtain gonad gametes and application - Google Patents
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Abstract
The invention discloses a method for inducing a young primate to obtain gonadal gametes. The method comprises the steps of administering gonadotropin releasing hormone or analogues thereof to female young primates, and administering gonadotropin in combination, inducing follicular development and obtaining mature oocytes, namely female gametes; the male young primate is given with gonadotropin and is given with gonadotropin releasing hormone or analogues thereof in combination to induce testicular spermatogenesis and obtain sperms which are male gametes; and (4) fertilizing the obtained female gamete and male gamete to enable the fertilized egg to develop into a blastocyst or a progeny. The combined hormone adopted by the invention can induce the sexual precocity of young primates, obtain the gonad gamete in advance, obviously shorten the generation cycle of the primates and accelerate the propagation of the offspring of the primates. The invention can realize rapid and large-scale propagation of young primates, shorten the generation period, reduce the cost and provide experience reference for conservation of precious and endangered animal germplasm resources and rapid propagation of gene editing animals.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for inducing a young primate to obtain gonadal gametes and application of the method.
Background
As is known, genetic information of a species is lost along with extinction, and in the present day that the number of endangered animals is increasing, how to safely and effectively obtain gonad gametes from young endangered animals and further obtain test-tube animals is a great problem for rescuing the endangered animals.
Primates have high homology with human genes and similar phylogeny, and are excellent experimental models for biomedical research. In recent years, important human diseases caused by gene mutation are more and more emphasized, and a primate transgenic model is generated. With the continuous innovation of gene editing technology, gene editing becomes easier and more efficient. However, the obtained transgenic primates are often heterozygotes, or the target gene sequences are different, and generation breeding is required to obtain transgenic primates with the same target gene sequence. Naturally, female cynomolgus monkeys can reach sexual maturity only by about 3 years old, male cynomolgus monkeys need longer time to reach sexual maturity generally until about 4.5 years old, and the cynomolgus monkeys have a longer gestation period (about 165 days) and are almost single-fetus, which determines that a long time is needed to obtain progeny transgenic primates.
Induction of sexual precocity in young animals is an important method for rapid propagation of offspring and preservation of species. The current research on sexual precocity mainly focuses on the treatment of female sexual precocity and the exploration of genes related to the sexual precocity. The hypothalamic-pituitary-gonadal axis (HPGA) is the fundamental mechanism for the development of precocious puberty. The hypothalamic-pituitary-gonadal axis is present during the formation of the reproductive system, is in an immature state until sexual maturity, and cannot promote gonadal development and the production of sexual gametes.
Currently, the only method available for obtaining sex-derived gametes from young primates is the xenotransplantation of testicular tissue (Liu et al, Generation of diseases with a special derivative from jraventile monkey biological tissues [ J ]. Cell Research,2016,26(1):139-142), but this method has significant drawbacks: (1) firstly, testicular excision of the young monkey is required, and the injury to the young monkey cannot be recovered and repeated; (2) the use time is long, and the sexual source gamete can be obtained after 12 months of transplantation; (3) monkeys presenting testicular tissue were older. At present, no relevant report is available on how to obtain gonad gametes of young animals in a short time and then obtain blastula or offspring of the young animals without causing damage to primates.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a method for inducing a young primate to obtain gonadal gametes. The method adopts a combined hormone method to realize the induction of sexual precocity of young primates, obtain sperms or mature oocytes of the primates and then obtain blastula or filial generations of the primates, thereby greatly shortening the generation cycle of the primates, accelerating the propagation of the descendants of the primates, overcoming the reproductive physiology phenomenon of insufficient development of neuroendocrine organs of young primates before sexual maturity, and particularly aiming at the rapid propagation of precious non-human primates and gene-edited young non-human primates. This not only induces sexual precocity in young primates, but also rapidly propagates their progeny, saving time and creating economic benefits.
The purpose of the invention is realized by the following technical scheme:
a method for inducing a young primate to obtain gonad gametes comprises the following specific steps:
s1, inducing female young primates: (1) administering a gonadotropin-releasing hormone or a gonadotropin-releasing hormone analogue to the female young primate daily until the end of the induction treatment; (2) when the level of the plasma estrogen is increased by 5-15 times, the follicle stimulating hormone is given at the same time; (3) on the day of cessation of the use of follitropin, gonadotropin is concurrently administered; (4) thereafter continuing administration of gonadotropin-releasing hormone or a gonadotropin-releasing hormone analogue to induce ovarian follicular development in the female young primate;
s2, taking follicular fluid from the female young primate induced in the step S1 to obtain an oocyte, namely a female gamete of the young primate;
s3, inducing male young primates: (1) administering gonadotropin to a young male primate, (2) stopping the administration of gonadotropin when the plasma testosterone level increases by 5-15 times or after 20-40 consecutive days, and starting the administration of gonadotropin-releasing hormone or a gonadotropin-releasing hormone analogue to induce spermatogenesis in the testis of the young male primate;
and S4, obtaining semen and sperms of the male young primate induced in the step S3, wherein the sperms are male gametes of the young primate.
In order to verify the developmental competence of the female gamete of the young primate described in step S2 and the male gamete of the young primate described in step S4, the female gamete and the male gamete are fertilized, and the fertilized egg develops to the blastocyst stage, and then the blastocyst or progeny thereof is obtained. It was also demonstrated in the examples that the combination hormone induced successful acquisition of mature gonadal gametes in young cynomolgus monkeys. Obviously, the method can obviously shorten the generation cycle of the primate and accelerate the propagation of the offspring.
Preferably, the gonadotropin-releasing hormone or the gonadotropin-releasing hormone analogue is administered in the step S1(1) in an amount of 0.1 to 50 μ g/kg/time based on the body weight of the young animal, and the frequency of administration of the gonadotropin-releasing hormone or the gonadotropin-releasing hormone analogue is 1 to 288 times/day for 15 to 60 consecutive days.
Preferably, in the step S1(2), the simultaneous administration time of the gonadotropin releasing hormone and the follitropin is 6-12 days, the administration amount of the follitropin is 5-35 IU/kg each time, and the administration frequency of the follitropin is 1-4 times/day; the gonadotropin-releasing hormone or gonadotropin-releasing hormone analogue is administered in the same amount and at the same frequency as in step S1 (1).
Preferably, the gonadotropin in step S1(3) is human chorionic gonadotropin or luteinizing hormone, and the gonadotropin is administered in an amount of 100 to 500 IU/kg based on the body weight of the young animal for only 1 time; the gonadotropin-releasing hormone or gonadotropin-releasing hormone analogue is administered in the same amount and at the same frequency as in step S1 (1); the amount and frequency of administration of the gonadotropin-releasing hormone or gonadotropin-releasing hormone analogue in step S1(4) are the same as those in step S1 (1).
Preferably, in step S3(1), the gonadotropin is luteinizing hormone, human chorionic gonadotropin or equine chorionic gonadotropin, each administration amount of the gonadotropin is 100 to 500 IU/kg based on the body weight of the young animal, and the administration frequency of the gonadotropin is 1 to 7 times/week.
Preferably, the dosage of the gonadotropin releasing hormone or the gonadotropin releasing hormone analogue in the step S3(2) is 0.1-50 microgram/kg based on the body weight of the young animal, the administration frequency of the gonadotropin releasing hormone or the gonadotropin releasing hormone analogue is 1-288 times/day, and the continuous administration is 15-120 days.
Preferably, the gonadotropin-releasing hormone analogue in steps S1(1) and S3(3) is gonadorelin, triptorelin or goserelin.
Preferably, the young primate of steps S1 and S3 is a primate from after weaning to the pre-sexual maturity stage.
More preferably, the primate is a rhesus monkey, a cynomolgus monkey, a ragtail monkey, a japanese monkey or a cynomolgus monkey.
The method is applied to the protection of non-human primates, gene editing young non-human primates and endangered animals.
The hypothalamic-pituitary-gonadal axis (HPGA) is the fundamental mechanism for the development of precocious puberty. The hypothalamic-pituitary-gonadal axis is present during the formation of the reproductive system, is under-developed state before sexual maturity, and cannot promote gonadal development and the production of sexual gametes. The gonadotropin releasing hormone (GnRH) used in the invention is secreted by hypothalamus, stimulates the secretion of pituitary gonadotropin, and induces the development of gonads. In the tissues of the reproductive organs, gonadotropin releasing hormone analogues (GnRHa) act systematically or similarly to GnRH, with some GnRHa acting more strongly or for a longer duration. Gonadotropins are glycoprotein hormones that regulate the development of the gonads of vertebrates and promote the production and secretion of sex hormones, such as luteinizing hormone (also known as luteinizing hormone, LH) and Follicle Stimulating Hormone (FSH) secreted from the anterior pituitary, which act synergistically to stimulate the development of germ cells in the ovary or testis and the production and secretion of sex hormones. Chorionic gonadotropin (hCG) secreted by human placenta has functions of FSH and LH, and stimulates the secretion of testosterone in fetal testis to promote male sexual differentiation before the secretion of LH by fetal pituitary; it also can promote gonad development, stimulate activity of mesenchymal cells in testis, and increase secretion of androgen (testosterone). Pregnant mare serum gonadotropin (also called mare chorionic gonadotropin) also has double activities similar to FSH and LH, is commonly used for replacing more expensive FSH in production to be widely applied to the oestrus induction, superovulation or ovulation rate increase of animals, and has the functions of promoting the development of sperm tubes and sexual cell differentiation of male animals.
The invention adopts a combined administration mode, in the female young primate, gonadotropin releasing hormone or gonadotropin releasing hormone analogue is administered, second reproductive hormone is continuously administered according to gonadal response, ovarian follicle development of the animal is jointly stimulated, and mature ova of the young primate are obtained by taking ova. For young male primates, motile sperm are obtained by artificial stimulation of semen collection by administering gonadotropins (equine chorionic gonadotropin, human chorionic gonadotropin or luteinizing growth hormone) and continuing administration of a second reproductive hormone in response to the gonadal response. The obtained ovum and sperm are gonad gametes of young primate.
Due to the similar reproductive system structure and function characteristics of rhesus monkeys, cynomolgus monkeys, ragtail monkeys, Japanese monkeys and cynomolgus monkeys, the reproductive characteristics such as menstrual cycles are very similar. Therefore, the invention can also be used for the research and protection of rhesus monkeys, cynomolgus monkeys, ragtail monkeys, Japanese monkeys or cynomolgus monkeys.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention adopts the method of combined hormone for the first time to realize the induction of the precocious puberty of the primate animal, obtains the sperms or the mature oocytes thereof, then obtains the blastocysts or the filial generations thereof, obviously shortens the generation cycle of the primate animal, accelerates the propagation of the filial generations thereof, and can be used for inducing the precocious puberty of the primate animal;
2. the method can obtain mature oocytes and active sperms of young primates in a large amount, quickly and repeatedly, shortens the gonad maturation period to 0.5-4 years, obviously shortens the generation period of the primates, saves time, improves the propagation efficiency, and effectively reduces the production cost or the experimental expenditure;
3. the invention provides experience reference for researches on primates such as endangered, precious and gene edited animals (gene mutation or transgenosis).
Drawings
FIG. 1 is a photograph of female and male gametes obtained from a combination hormone-treated young monkey.
Figure 2 is a graph of the change in serum testosterone levels in male puppy before and after combined hormone treatment.
Detailed Description
The following examples are presented to further illustrate the present invention and should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
In the embodiment, the cynomolgus monkeys are provided by Guangdong island biotechnology limited, and are free of specific pathogens through detection, and the weight of the cynomolgus monkeys is about 1-3 kg.
Example 1
Gonadotropin releasing hormone analogue (gonadorelin) is combined with human follicle stimulating hormone and human chorionic gonadotropin to induce young female monkeys to obtain gonadal gametes.
1. A female cynomolgus monkey of 1 year old is taken, the weight of the female cynomolgus monkey is 1.0 kg, and no premature sexual maturity symptoms exist, for example, the level of estradiol in blood plasma is 20ng/mL, and no abnormality exists in other physiological aspects.
2. Gonadorelin was injected daily at 28.8 micrograms, dissolved in physiological saline and divided into 288 aliquots, injected subcutaneously 1 time 5 minutes apart for 60 days.
3. After the injection of gonadorelin is started, blood is drawn once a week, the plasma estrogen level is detected, and the gradual increase of the sexual skin range of the young monkey and the gradual change of the color into bright red can be observed, which indicates that the plasma estrogen level is increased and the female young monkey gradually reaches sexual maturity. And the estrogen concentration was measured to rise from the initial 20ng/mL and reach 80 ng/mL.
4. And (2) continuing to inject gonadorelin, simultaneously starting to inject 5IU of recombinant human follicle-stimulating hormone 4 times a day, continuously injecting for 12 days, after stopping injecting the recombinant human follicle-stimulating hormone, injecting 500IU of human chorionic gonadotropin for 1 time, continuing to inject gonadorelin with the same dosage as that in the step 2, observing ovaries and sucking follicular fluid by means of a laparoscope within 35-36 hours later, and simultaneously ending the injection.
5. And observing the oocyte in the follicular fluid under a stereoscopic microscope, wherein the mature oocyte is obtained when the first polar body appears in the oocyte. Obtaining 22 oocytes, wherein 15 mature oocytes (shown in figure 1) are female gametes of the young primate.
6. The fertilization method of single sperm injection is utilized to fertilize mature oocytes, fertilized eggs are cultured by the conventional in-vitro embryo culture method, the fertilized eggs develop to blastocysts, and the blastocyst development rate reaches 40%.
In FIG. 1, (a) shows mature oocytes obtained by treating female young monkeys with a combination hormone. The mature oocytes showing the first polar body can be seen in FIG. 1 (a). The sex gland gamete is successfully obtained by inducing young female cynomolgus monkeys by the combined hormone.
Example 2
The gonadal gametes are obtained by inducing young male cynomolgus monkeys to acquire human chorionic gonadotropin in combination with a gonadotropin releasing hormone analogue (gonadorelin).
1. A male cynomolgus monkey of 2 years old is taken, the weight of the male cynomolgus monkey is 1.6 kg, and no premature sexual maturity symptoms exist, for example, the testosterone level of blood plasma is less than 1.0ng/mL, and other physiological aspects are not abnormal.
2. 160IU equine chorionic gonadotropin was injected 1 time a day for 15 consecutive days. Plasma testosterone levels up to 5.0ng/mL were measured by blood draw one week prior to the start of equine chorionic gonadotropin and once a week after injection.
3. Then 60 mug of gonadorelin, a gonadotropin releasing hormone analogue, was injected daily, dissolved in physiological saline and divided into six equal portions, and the injections were administered by intramuscular injection 6 times at 3 hour intervals every day for 90 days.
4. And (3) continuing to administer gonadorelin with the same dose and the same method in the step 3, and simultaneously stimulating the rectum or penis of the young monkey by utilizing an artificial induced semen collection method to induce the young monkey to discharge semen so as to obtain the semen and the normal moving live sperm, namely the young male gamete of the primate (shown in the figure 1 b). The artificial induction semen collection method and semen collection can be repeated at intervals of 1 week.
5. The obtained sperms are used for injecting the sperms to fertilize mature oocytes, fertilized eggs are cultured by a conventional in-vitro embryo culture method, the fertilized eggs develop to blastocysts, and the blastocyst development rate reaches 50%.
In FIG. 1, (b) shows mature sperm obtained by treating male young monkeys with a combination hormone. The fully structured mature sperm can be seen in FIG. 1. The sex gland gamete is successfully obtained by inducing the young male cynomolgus monkey with the combined hormone. Figure 2 is a graph of the change in serum testosterone levels in male puppy before and after combined hormone treatment. From figure 2 it can be seen that the young monkeys have significantly elevated testosterone levels after combined hormone treatment. It shows that the male young monkey can respond to combined hormone stimulation quickly, and the male young monkey gradually reaches sexual maturity (the rise of the serum testosterone level is an important index of male sexual maturity).
Example 3
The gonadal gametes are obtained by inducing young male cynomolgus monkeys to acquire human chorionic gonadotropin in combination with a gonadotropin releasing hormone analogue (gonadorelin).
1. 3.5kg of a male cynomolgus monkey of 3 years old is taken, and has no premature maturation symptoms, for example, the testosterone level in blood plasma is less than 1.0ng/mL, and other physiological aspects are not abnormal.
2. 1750IU equine chorionic gonadotropin was injected 1 time per week for 3 weeks. Plasma testosterone levels above 10.0ng/mL were measured by blood draw one week prior to the start of equine chorionic gonadotropin and once a week after injection.
3. Subsequently, 80. mu.g of gonadorelin was injected daily, dissolved in physiological saline, divided into 4 equal portions, and injected intramuscularly 4 times at 6-hour intervals daily for 15 days.
4. And (3) continuously administering gonadorelin with the same dose and the same method in the step 3, and simultaneously stimulating the rectum or penis of the young monkey by utilizing an artificial induction semen collection method to induce the young monkey to discharge semen so as to obtain semen and normal-movement live sperm, namely male gametes of the young primate. The artificial induction semen collection method and semen collection can be repeated at intervals of 1 week.
Example 4
Human chorionic gonadotropin in combination with gonadotropin releasing hormone analogue (gonadorelin) induces young male rhesus monkeys to acquire gonadal gametes.
1. A male rhesus monkey young monkey of 1 year old is taken, the weight of the male rhesus monkey young monkey is 1.5 kg, and no premature sexual maturity symptoms exist, such as the testosterone level of blood plasma is less than 1.0ng/mL, and no abnormality exists in other physiological aspects.
2. 200IU human chorionic gonadotropin is injected 1 time a day for 30 days. Plasma testosterone levels above 5.0ng/mL were measured by blood draw one week prior to the start of human chorionic gonadotropin and once per week after injection.
3. Then, 40. mu.g of gonadorelin was injected daily, dissolved in physiological saline, and divided into 9 equal portions, and the injections were administered by intramuscular injection 9 times at 2-hour intervals every day for 120 days.
4. And (3) continuously administering gonadorelin with the same dose and the same method in the step 3, and simultaneously stimulating the rectum or penis of the young monkey by utilizing an artificial induction semen collection method to induce the young monkey to discharge semen so as to obtain semen and normal-movement live sperm, namely male gametes of the young primate. The artificial induction semen collection method and semen collection can be repeated at intervals of 1 week.
Example 5
Gonadotropin releasing hormone analogue (triptorelin) is combined with human follicle stimulating hormone and human chorionic gonadotropin to induce young female monkeys to obtain gonadal gametes.
1. A female cynomolgus monkey of 2.5 years old is taken, the weight of the female cynomolgus monkey is 2.5 kg, no premature sexual maturity symptoms exist, for example, the level of estradiol in blood plasma is lower than 20ng/mL, and no abnormality exists in other physiological aspects.
2. The dosage of triptorelin is 125 micrograms per day, and the triptorelin is dissolved in physiological saline and injected for 1 time per day for 15 days.
3. After the start of triptorelin injection, blood was drawn weekly and plasma estrogen levels were measured. And the estrogen concentration was measured to rise from initially below 20ng/mL to 80 ng/mL.
4. Continuing to inject triptorelin and simultaneously beginning to inject 87.5IU of recombinant human follicle-stimulating hormone 1 time a day for 6 days continuously, after stopping injecting the recombinant human follicle-stimulating hormone, injecting 1250IU of human chorionic gonadotropin 1 time, continuing to inject the same dose of triptorelin in the step 3, and observing the ovary and sucking follicular fluid by means of a laparoscope within 32-36 hours later, and simultaneously ending the injection.
5. And (5) picking up the oocyte in the follicular fluid under a stereoscopic microscope.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations and simplifications are intended to be included in the scope of the present invention.
Claims (2)
1. A method for inducing a young primate to obtain gonadal gametes is characterized by comprising the following specific steps:
s1, inducing female young primates: the primate is a rhesus monkey, a cynomolgus monkey, a ragtail monkey, a Japanese monkey or a cynomolgus monkey; (1) administering a gonadotropin-releasing hormone or a gonadotropin-releasing hormone analogue to the female young primate daily until the end of the induction treatment; the administration amount of the gonadotropin releasing hormone or the gonadotropin releasing hormone analogue is 28.8-50 micrograms/kg/day according to the body weight of the young animal, and the continuous administration is carried out for 15-60 days; (2) when the level of the plasma estrogen is increased by 4-5 times, the follicle stimulating hormone is given at the same time; the simultaneous administration time of the gonadotropin releasing hormone and the follicle stimulating hormone is 6-12 days, and the daily administration amount of the follicle stimulating hormone is 20-35 IU/kg; (3) on the day of cessation of the use of follitropin, gonadotropin is concurrently administered; the gonadotropin is human chorionic gonadotropin or luteinizing hormone, and the administration amount of the gonadotropin is 500 IU/kg based on the weight of the young animal, and the administration is only 1 time; (4) thereafter continuing administration of gonadotropin-releasing hormone or a gonadotropin-releasing hormone analogue to induce ovarian follicular development in the female young primate; wherein the administration amounts of the gonadotropin releasing hormone or the gonadotropin releasing hormone analogue in (1) to (4) are respectively 28.8 to 50 micrograms/kg/day according to the body weight of the young animal, and the continuous administration is carried out for 15 to 60 days; the gonadotropin releasing hormone analogue is gonadorelin, triptorelin or goserelin;
s2, taking follicular fluid from the female young primate induced in the step S1 to obtain an oocyte, namely a female gamete of the young primate;
s3, inducing male young primates: (1) the method comprises the following steps of (1) administering gonadotropin to a male young primate, wherein the gonadotropin is luteinizing hormone, human chorionic gonadotropin or equine chorionic gonadotropin, the administration amount of the gonadotropin is 100-500 IU/kg/day according to the body weight of the young primate, and the administration frequency of the gonadotropin is 1-7 times/week; (2) when the level of testosterone in blood plasma is increased by 5-10 times or continuously administered for 20-40 days, the administration of gonadotropin is stopped, and the administration of gonadotropin-releasing hormone or gonadotropin-releasing hormone analogue is started to induce the spermatogenesis of testicles of young males; the gonadotropin releasing hormone analogue is gonadorelin, triptorelin or goserelin; the administration amount of the gonadotropin releasing hormone or the gonadotropin releasing hormone analogue is 22.9-37.5 micrograms/kg/day according to the body weight of the young animals, and the administration time is 15-120 days respectively;
s4, obtaining semen and sperms of the male young primates induced in the step S3, wherein the sperms are male gametes of the young primates; the gonad maturation period of the young primate is shortened to 0.5-4 years.
2. The method for inducing a juvenile primate to acquire gonads gametes according to claim 1, wherein the juvenile primate is a primate from after weaning to a pre-sexual maturity stage in steps S1 and S3.
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