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CN-121991885-A - Culture medium for in-vitro induced differentiation of flounder spermatogonial stem cells and application

CN121991885ACN 121991885 ACN121991885 ACN 121991885ACN-121991885-A

Abstract

The invention belongs to the technical field of biology, and particularly relates to a culture medium for in-vitro induced differentiation of bastard halibut spermatogonial stem cells and application thereof. According to the invention, by optimizing the formula of the induced differentiation medium, combining specific growth factors and hormones, and establishing a step-by-step collection and artificial insemination procedure, the directional differentiation of long-term passage spermatogenic stem cells to sperms with insemination capability is successfully realized, and the normal development of paralichthys olivaceus filial generation is finally obtained. The method breaks through the key technical bottleneck that sperm function is incomplete after long-term passage spermatogonial stem cell induced differentiation and viable offspring cannot be obtained, and provides a reliable technical platform for the preservation of paralichthys olivaceus germplasm resources, genetic breeding acceleration and reproductive development research.

Inventors

  • REN YUQIN
  • HAN TIAN
  • CAO WEI
  • HOU JILUN
  • WANG XIYUAN
  • YANG YUCONG
  • WANG GUIXING
  • SAN LIZE
  • ZHANG HENG
  • LIU MINGYANG
  • Li Bingbu

Assignees

  • 中国水产科学研究院北戴河中心实验站

Dates

Publication Date
20260508
Application Date
20260210

Claims (9)

  1. 1. A culture medium for in vitro induced differentiation of flounder spermatogonial stem cells, which is characterized by comprising the following components: the basal medium was L-15, 15% FBS, 1% fish serum, 1% embryo extract protein 、bFGF 2 ng/ml、LIF 2 ng/ml、GDNF 40 ng/ml、SCF 100 ng/ml、EGF 10 ng/ml、11-KT 10~100 ng/ml、DHP 10 ng/ml、HCG 1 IU/ml、RA 1.5 ng/ml and melatonin 2.3 μg/ml.
  2. 2. An in vitro directional differentiation method for long-term passage of paralichthys olivaceus spermatogonial stem cells is characterized by comprising the following steps of: After the seminoma stem cells passaged for a long time of paralichthys olivaceus are cultured to a full culture flask, replacing the culture medium with the culture medium according to claim 1, and replacing half of the culture medium every 3-4 days at 23 ℃; and after 10-21 days of induced differentiation, collecting sperms in the culture medium.
  3. 3. The method for in vitro directed differentiation according to claim 2, wherein the age of the paralichthys olivaceus is less than or equal to 1 age.
  4. 4. The method for in vitro directed differentiation according to claim 2, wherein said method for collecting sperm is: (1) Collecting suspended sperms in the culture medium, and collecting the 5 ml culture medium in a 15 ml centrifuge tube; (2) Collecting adherent sperms in a culture medium, adding 1ml of pancreatin with the concentration of 0.05% into a culture bottle, shaking for 3-4 times to obtain partial adherent sperms, and neutralizing pancreatin by using an L-15 culture medium containing 15% FBS to obtain suspension containing the rest adherent sperms; (3) Combining the suspended sperm with a suspension comprising the remaining adherent sperm to obtain a complete sperm suspension; (4) Centrifuging the complete sperm suspension at 1500rpm, taking the sediment, discarding the supernatant, and supplementing the sediment with subculture solution to obtain the sperm to be subjected to artificial insemination.
  5. 5. A method of artificial insemination of paralichthys olivaceus, the method comprising the steps of: And (3) collecting well-developed female roe 0.5 ml, transferring the well-developed female roe into a culture dish with sperms to be subjected to artificial insemination, adding 4 ml seawater to the culture dish to activate the sperms, and completing artificial insemination of the paralichthys olivaceus after 1 h.
  6. 6. A paralichthys olivaceus embryo, characterized in that it is obtained by the artificial insemination method according to claim 5.
  7. 7. The culture medium of claim 1, the in vitro directional differentiation method of any one of claims 2-4, the artificial insemination method of claim 5, and the application of the paralichthys olivaceus embryo of claim 6 in preservation of paralichthys olivaceus germplasm resources.
  8. 8. The culture medium of claim 1, the in vitro directional differentiation method of any one of claims 2-4, the artificial insemination method of claim 5, and the use of the paralichthys olivaceus embryo of claim 6 in accelerating the breeding cycle of paralichthys olivaceus.
  9. 9. The culture medium of claim 1, the in vitro directional differentiation method of any one of claims 2-4, the artificial insemination method of claim 5, and the application of the paralichthys olivaceus embryo of claim 6 in research on reproductive development mechanism of paralichthys olivaceus.

Description

Culture medium for in-vitro induced differentiation of flounder spermatogonial stem cells and application Technical Field The invention belongs to the technical field of biology, and particularly relates to a culture medium for in-vitro induced differentiation of bastard halibut spermatogonial stem cells and application thereof. Background Fish spermatogonial stem cells are male germ stem cells that can transfer genetic resources to the next generation. Once the spermatogenic stem cell line is established, a large number of germ stem cells are obtained within a certain period of time, and the purpose of high-efficiency preservation of germ plasm resources of the male fish can be achieved through an in-vitro induced differentiation technology and a germ stem cell transplanting technology. However, the germ stem cell transplantation technology has certain limitations, time is required for preparing sterile receptors, the receptors need longer time to breed, risks such as diseases exist, and the like are restrictive factors such as high storage cost of the surrogate receptors. Once the in vitro induced differentiation technology breaks through, the process from spermatogonial stem cells to sperms can be realized only by short treatment in a laboratory within one month, and the spermatogenesis process can be completed without a propagation period like a living body, so that the development process is greatly accelerated, a research platform is provided for a germ cell development molecule regulation mechanism, and a powerful technical support is provided for protecting fish germplasm resources and even accelerating a variety breeding period. In the aspect of fish spermatogenic stem cell induced differentiation, aquatic product related specialists develop research on the in vitro induced differentiation of primary spermatogenic stem cells of zebra fish, clarias fuscus, gobio maculatus, japanese eel and Chinese black-tail, and the methods of in vitro induced differentiation, sperm collection and artificial insemination are not suitable for long-term passage spermatogenic stem cell lines, and can not lead the induced differentiation of long-term passage spermatogenic stem cells to generate survival filial generation. Whereas established spermatogenic cell lines of medaka, malakou, grouper, takifugu rubripes, alosa sapidissima, coilia ectenes, long-term passage of silurus meridionalis, although induced to differentiate, produced sperm-like cells, failed to produce viable offspring. Disclosure of Invention Based on the requirement, the invention provides a culture medium for in-vitro induced differentiation of bastard halibut spermatogonial stem cells and related application thereof, which can stably and simply carry out the treatments of in-vitro induced differentiation, collection, artificial insemination and the like of the bastard halibut spermatogonial stem cells for long-term passage, effectively solve the problem that the induced differentiation of the bastard halibut can not produce filial generation, and provide powerful technical support for the development of the germ stem cells of the bastard halibut, the efficient preservation and utilization of germplasm resources and the acceleration of the breeding process of new varieties. In order to achieve the above object, the present invention is realized by the following means: the invention provides a culture medium for in-vitro induced differentiation of flounder spermatogonial stem cells, which consists of the following components: The basal medium is L-15, 15% FBS, 1% fish serum (v/v) and 1% embryo extracted protein (v/v), after collecting and cleaning fertilized eggs of paralichthys olivaceus which are about to come out of the membrane for many times, adding an L-5 culture solution, homogenizing by a homogenizer, repeatedly freezing and thawing the homogenate for 3 times, centrifuging at 13000 rpm and 4 ℃ for 30 min, discarding supernatant, diluting with L-15 according to a ratio of 1:1, sterilizing and filtering by a 0.22 mu m filter, measuring protein concentration, and finally fixing the volume to a concentration ）、bFGF 2 ng/ml、LIF 2 ng/ml、GDNF 40 ng/ml、SCF 100 ng/ml、EGF 10 ng/ml、11-KT 10~100 ng/ml、DHP 10 ng/ml、HCG 1 IU/ml、RA 1.5 ng/ml of 18 g/L and a concentration ）、bFGF 2 ng/ml、LIF 2 ng/ml、GDNF 40 ng/ml、SCF 100 ng/ml、EGF 10 ng/ml、11-KT 10~100 ng/ml、DHP 10 ng/ml、HCG 1 IU/ml、RA 1.5 ng/ml of melatonin of 2.3 mu g/ml. The invention also provides an in vitro directional differentiation method of long-term passage seminoma stem cells of paralichthys olivaceus, which comprises the following steps: After the long-term passage of the paralichthys olivaceus is carried out until the seminiferous stem cells grow up to a culture bottle, replacing the culture medium with the culture medium, and replacing half of the culture medium every 3-4 days at the temperature of 23 ℃; and after 10-21 days of induced differentiation, collecting sperms in the culture medium. In