CN112048468A

CN112048468A - In-vitro maturation culture reagent for oocyte

Info

Publication number: CN112048468A
Application number: CN202010798991.8A
Authority: CN
Inventors: 凌秀凤; 赵纯; 霍然; 杨烨; 张军强; 于晓宁; 曹善仁
Original assignee: Nanjing Maternity and Child Healthcare Hospital
Current assignee: Nanjing Maternity and Child Healthcare Hospital
Priority date: 2019-09-03
Filing date: 2019-09-03
Publication date: 2020-12-08
Anticipated expiration: 2039-09-03
Also published as: CN110669123B; CN112048017A; CN112048017B; CN110669123A; CN112048468B

Abstract

The invention discloses an oocyte in-vitro maturation culture reagent. An in vitro maturation culture reagent for oocyte, which comprises a secretory endogenous polypeptide PDFF-CO3 with an amino acid sequence shown as SEQ ID NO. 1. PDFF-CO3 is added into the egg cell culture solution in the GV stage of the mouse, so that the biological function of promoting the maturation of the egg cells is realized; therefore, PDFF-CO3 can be applied to preparation of medicaments for promoting egg cell maturation and improving egg cell development potential and preparation of culture solutions.

Description

In-vitro maturation culture reagent for oocyte

Description of the cases

The invention relates to a divisional application of a secreted endogenous polypeptide PDFF-CO3 and a Chinese invention patent application with the application date of 2019-9-3 and the application number of 2019108274356.

Technical Field

The invention belongs to the technical field of assisted reproduction, and relates to a secretory endogenous polypeptide PDFF-CO3 and application thereof.

Background

In vitro maturation of oocytes (IVM) is a technique in which immature oocytes are obtained from ovaries without superovulation or after the application of a small amount of Gn, and cultured under in vitro adapted conditions to mature the oocytes and to have fertilization ability. As a hotspot frontier technology for assisted fertility, although the technology has wide application prospects in the fields of treatment of polycystic ovarian syndrome (PCOS), high/low ovarian response, uneven ovarian response, egg supply, female fertility preservation and the like, relevant research statistics show that the current oocyte in-vitro maturation culture system is not complete, the maturation rate is low, the development potential is poor and the like, the current clinical pregnancy rate is only 20-35%, and the technology becomes a main bottleneck of clinical application. Therefore, the improvement of the IVM success rate is the key of research in the technical field of assisted reproduction in recent years. In oocyte in vitro maturation studies, the composition of IVM medium directly affects the maturation rate of oocytes and subsequent cleavage and embryo development. Through years of research, scholars at home and abroad gradually discover that an IVM culture system is continuously optimized and the culture effect is improved by adding gonadotropin, steroid hormone, antioxidant, meiosis inhibitor, growth factor and the like into an IVM culture solution, but the effects of improving the maturation rate of oocytes and improving the development potential of the oocytes at present are not satisfactory. The search for safe and effective IVM culture solution additive components to promote the oocyte in vitro maturation and development potential is an important research direction at present.

Changes in the content of certain proteins or peptides in follicular fluid or changes in modifications can precisely reflect the physiological process of follicular development and maturation, and thus follicular fluid becomes a unique window for studying follicular development. However, since the follicular fluid contains complex components, not only contains ideal components for promoting oocyte maturation, but also contains substances for inhibiting oocyte maturation, such as follistatin, maturation inhibition factor, and the like, which are not beneficial to research on the influence of single factors on oocyte in vitro maturation, the identification of factors for promoting oocyte maturation and improving oocyte development potential in follicular fluid to optimize an IVM culture system may bring more surprise to the people. In recent years, the function of small molecule polypeptides in organisms has been receiving more and more attention, and they are involved in regulating various physiological and pathological processes in the human body, such as: the polypeptide regulates the secretion of cells by regulating the secretion of auxin and influences the growth, differentiation, apoptosis and the like of the cells by regulating various signal paths. In recent years, the function of small molecule polypeptides in oocyte maturation has been of common interest to reproductive researchers, such as: zhang M and the like find that C-type natriuretic peptide (CNP) is a key substance for maintaining meiotic block of oocytes in follicles, and the CNP is added into in-vitro maturation culture solutions of ova of different mammals such as mice, cows, goats, pigs and the like, so that the maturation of the ova can be promoted, and the in-vitro fertilization rate and the blastocyst formation rate can be improved. Because: the research finds that some endogenous polypeptides naturally produced by cells have secretion polarity and can be secreted into body fluid to participate in the occurrence and development of diseases; secondly, the change of the content of some key polypeptides in the follicular fluid can predict the development potential of the oocyte; the micromolecular polypeptide has the advantages of strong stability, small toxic and side effects, high specificity and the like, and is widely applied to clinical drug development, so that a new research clue is provided for people to explore and improve the components of the IVM culture medium from the perspective of searching secreted polypeptide in follicular fluid.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide a secreted endogenous polypeptide PDFF-CO 3.

The invention also aims to provide application of the secreted endogenous polypeptide PDFF-CO 3.

The purpose of the invention can be realized by the following technical scheme:

a secreted endogenous polypeptide PDFF-CO3 is characterized by having an amino acid sequence represented by SVQLTEKRMDKVGKYPKELRKCCED (SEQ ID NO. 1).

The secretory endogenous polypeptide PDFF-CO3 is applied to in-vitro maturation culture of oocytes.

The invention relates to application of a secretory endogenous polypeptide PDFF-CO3 in preparation of an oocyte in-vitro maturation culture reagent.

An oocyte in-vitro maturation culture reagent, which comprises the secreted endogenous polypeptide PDFF-CO 3.

As a preferable selection of the invention, the concentration of the secretory endogenous polypeptide PDFF-CO3 in the oocyte in-vitro maturation culture reagent is 1-2 mu M, and 1.5 mu M is preferable.

The invention discloses an oocyte in-vitro maturation culture method, which is characterized in that an oocyte in-vitro maturation culture reagent disclosed by the invention is used for carrying out in-vitro culture on an oocyte, or a secretory endogenous polypeptide PDFF-CO3 disclosed by the invention is added into an oocyte in-vitro maturation culture solution.

As a preferable selection of the invention, the secretory endogenous polypeptide PDFF-CO3 with the concentration of 1-2 mu M can be added into an oocyte in-vitro maturation culture solution.

As a further preferred aspect of the present invention, the secreted endogenous polypeptide PDFF-CO3 can be added to the oocyte in vitro maturation medium at a concentration of 1.5. mu.M.

Has the advantages that:

the embryo secretory endogenous polypeptide PDFF-CO3(SVQLTEKRMDKVGKYPKELRKCCED) is a CO3 source peptide, consists of 25 amino acids, and has no related function report at present. Bioinformatics analysis and experimental results show that the PDFF-CO3 is derived from the CO3 protein chainA, and the region is also present on CO3 proteins of mice, cows, pigs and the like, so that the sequence of PDFF-CO3 is highly conserved in mammals. ProtParam on-line analysis shows that the Instability coefficient (Instability index) of PDFF-CO3 is 37.1, belongs to stable polypeptide and can exist stably; the fat index (Aliphatic index) was 54.4 and the average hydrophilicity and hydrophobicity (Grand average of hydropathicity) was-1.256. And thirdly, adding FITC-marked PDFF-CO3 into the culture solution of the egg cells in the GV stage of the mouse, and observing and finding that the fluorescence is distributed in perivitelline space and cells of the egg cells in the MII stage by laser confocal observation after 14 hours, thereby indicating that PDFF-CO3 can penetrate the zona pellucida and the cell membranes of the embryo to play a role. PDFF-CO3 is added into the egg cell culture solution in the GV stage of the mouse, so that the biological function of promoting the maturation of the egg cells is realized; therefore, PDFF-CO3 can be applied to preparation of medicaments for promoting egg cell maturation and improving egg cell development potential and preparation of culture solutions.

Drawings

FIG. 1 the PDFF-CO3 sequence is highly conserved in various animals.

FIG. 2 the three-dimensional structures of human and mouse CO3 proteins share high homology.

FIG. 3 the CO3 protein domain is highly conserved in various animals.

FIG. 4 location of FITC-labeled PDFF-CO3 in the egg cells after 14 hours of incubation in mouse GV-stage egg cell culture fluid.

FIG. 5 the effect of different concentrations of PDFF-CO3 on the in vitro maturation of mouse GV stage egg cells.

FIG. 6A. Effect of different concentrations of PDFF-CO3 on the rupture of mouse GV stage egg foaming. B. Effect of different concentrations of PDFF-CO3 on in vitro maturation of mouse GV stage egg cells.

Detailed Description

Example 1

To eliminate individual differences, 5 young IVF patients (patients treated with IVF due to oviduct factors, age ≤ 30 years, menstruation regulation, basal endocrine, AMH normal) were collected respectively for their mature follicular fluid (mature group) and antral follicular fluid (immature group of follicles 1.0-1.2cm in diameter, which develop asynchronously during superovulation using the antagonist protocol and need to be punctured in advance), and subjected to polypeptide difference analysis using proteomics. Through mass spectrum identification, 519 polypeptides are totally found in the follicular fluid, wherein 311 polypeptides are significantly and differentially expressed between two groups (the difference multiple is more than or equal to 2, T-test is carried out, and P is less than 0.05).

Example 2

The NCBI database displays: PDFF-CO3 is derived from the CO3 protein chainA,

(https:// www.ncbi.nlm.nih.gov/protein/4I6O _ a. This region is also present in the CO3 protein of mouse, cow, pig, etc., so the sequence of PDFF-CO3 is highly conserved in mammals (FIG. 1). ProtParam on-line analysis shows that the Instability coefficient (Instability index) of PDFF-CO3 is 37.1, belongs to stable polypeptide and can exist stably; the fat index (Aliphatic index) was 54.4 and the average hydrophilicity and hydrophobicity (Grand average of hydropathicity) was-1.256.

(https:// web. expasy. org/cgi-bin/protparam/protparam.) the three-dimensional structure of human and mouse CO3 proteins was predicted by pymol software and found to be strikingly highly homologous (FIG. 2); online databases also show that the CO3 protein domain is highly conserved across multiple animals (fig. 3).

Example 3

FITC fluorescent labeled peptide fragment of PDFF-CO3 is synthesized by chemical synthesis method, added into M16 culture solution (1.5 mu M), cultured for 14h, collected in MII stage egg cells, and then stained with hoechst (1:500) for 10 min. Fluorescence was observed by confocal laser observation and distributed in perivitelline space and cells (FIG. 4), indicating that PDFF-CO3 can pass through the zona pellucida and cell membrane of embryo to function.

Example 4

Synthesizing a peptide derived from CO3, namely PDFF-CO3 by adopting a chemical synthesis method, wherein the amino acid sequence is shown as SEQ ID NO.1, culturing the egg cells at the GV stage of a mouse, and adding PDBCM polypeptides with different concentrations (1.5nM, 1.5 mu M and 1.5mM) into an embryo culture solution (M16) to stimulate: taking 3-week ICR female mouse ovary, obtaining GV-stage egg cells under microscope, washing naked eggs in-vitro operating fluid M2, placing at 37 deg.C before 5% CO₂Well balanced and added with PDBCM of different concentrations in M16 culture solution. The development and proportion of GVBD and MII were observed and counted at 3 and 14 hours. The influence of the polypeptide on the in vitro maturation of the mouse GV-stage egg cells is observed, and the result shows that the in vitro maturation rate of the mouse GV-stage egg cells can be remarkably improved by adding 1.5 mu M of polypeptide PDBCM (figures 5 and 6).

Sequence listing

<110> Nanjing City health care hospital for women and children

<120> in vitro maturation culture reagent for oocyte

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 25

<212> PRT

<213> human (amines)

<400> 1

Ser Val Gln Leu Thr Glu Lys Arg Met Asp Lys Val Gly Lys Tyr Pro

1 5 10 15

Lys Glu Leu Arg Lys Cys Cys Glu Asp

20 25

Claims

1. An in vitro maturation culture reagent for oocyte, which is characterized by comprising a secretory endogenous polypeptide PDFF-CO3 with an amino acid sequence shown as SEQ ID NO. 1.

2. The oocyte in-vitro maturation culture reagent according to claim 1, wherein the concentration of the secreted endogenous polypeptide PDFF-CO3 is 1-2 μ M.

3. The oocyte in vitro maturation culture reagent according to claim 2, characterized in that the concentration of the secreted endogenous polypeptide PDFF-CO3 is 1.5 μ M.