CN115873788A

CN115873788A - Application of OOSP2 protein in promotion of egg development

Info

Publication number: CN115873788A
Application number: CN202111129856.5A
Authority: CN
Inventors: 纪家葵; 胡文奇
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2021-09-26
Filing date: 2021-09-26
Publication date: 2023-03-31

Abstract

The invention discloses an application of OOSP2 protein in promoting ovum development. The invention provides an application of human OOSP2 protein or related biological materials thereof in any one of the following applications: promoting the development of ova and inducing the maturation of oocytes. The result of the invention shows that the human OOSP2 protein can effectively induce the maturation of oocytes in vitro, and can improve the utilization rate of ova and the pregnancy outcome in the test tube infant cycle. For patients with low egg maturation rates, such as patients with polycystic ovary syndrome, gonadotropin-stimulated high-response patients, ovarian low-response patients, etc., these patients cannot obtain enough mature eggs through the conventional ovulation promoting protocol. By utilizing the human OOSP2 protein, the in vitro maturation rate of the ovum can be effectively improved, the utilization rate of the ovum of patients can be improved, and the pregnancy outcome can be improved. Has important significance in IVF clinical application.

Description

Application of OOSP2 protein in promotion of egg development

Technical Field

The invention relates to the field of biotechnology and medicine, in particular to application of OOSP2 protein in promoting ovum development.

Background

OOSP2 protein, all known as Ocyte-secreted protein 2, also known as TMEM122, PLAC1L. Is a secreted protein (Wagner, M., yoshihara, M., douagi, I., damdemopoulos, A., panula, S., petropoulos, S., lu, H., pettersson, K., palm, K., katayama, S., et al, (2020). Single-cell analysis of human over design identification cell deposition but not yet, 17.971KD, with 158 amino acids, located on human chromosome 11) highly expressed in human egg cells.

At embryonic stage or just birth, the oocyte enters the first meiotic prophase to become a primary oocyte, develops through the fine line phase, the even line phase and the pachytene phase and stagnates in the nucleus net phase divided after the double line phase. The obvious characteristic of oocytes at this stage is a large nucleus, called the Germinal Vesicle (GV). Until after sexual maturation, under the action of gonadotropins or other factors (e.g., epidermal growth factor), the oocyte can resume meiosis, a process also known as oocyte capacitation (capacitation), which includes nuclear resumption of meiosis to metaphase (M) quiescence of second meiosis and maturation of the cytoplasm that undergoes a series of complex physiological and biochemical changes.

At present, no report about the promotion of the development of the ovum by the human OOSP2 exists.

Disclosure of Invention

The invention aims to provide a new application of OOSP2 protein.

In a first aspect, the invention claims the use of a human OOSP2 protein or related biological material thereof in any of:

p1, promoting the development of ova;

p2, preparing a product for promoting the development of ova;

p3, inducing oocyte maturation;

p4, preparing a product for inducing oocyte maturation.

The related biological material is a nucleic acid molecule capable of expressing the human OOSP2 protein or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule, or a substance capable of promoting the expression of the human OOSP2 protein and/or increasing the activity of the human OOSP2 protein.

Wherein the promotion of ovum development is the promotion of ovum development in vitro. The induced oocyte maturation is in vitro induced oocyte maturation.

In a second aspect, the invention claims the use of a human OOSP2 protein or its related biological material in any of the following:

q1, improving the oocyte maturation rate;

q2, preparing a product for improving the maturation rate of the oocyte;

q3, improving the maturation rate of the oocyte;

q4, preparing a product for improving the maturation rate of the oocyte.

Wherein, the increasing of the oocyte maturation rate is increasing of the oocyte maturation rate in vitro; the increasing of the oocyte maturation rate is an in vitro increasing of the oocyte maturation rate.

In a third aspect, the invention claims a method for inducing oocyte maturation in vitro.

The method for inducing oocyte maturation in vitro as claimed in the present invention may include the following steps: immature oocytes (GV) were cultured in a medium containing human OOSP2 protein at a final concentration of 150ng/mL to thereby obtain mature oocytes.

In a particular embodiment of the invention, the medium is an IVF medium. The culture medium is covered with a layer of mineral oil. In the method, each oocyte is cultured separately in a separate chamber in an embryoside dish (vitrolite). The culture conditions were 37 ℃,6% CO ₂ . The culture time is 72h.

In addition, the methods can be used to increase oocyte maturation (rate) rates in vitro, as well as to promote egg development in vitro.

In the present invention, the oocyte maturation is embodied in the form of blastocyst disruption (GVBD) and/or Polar Body Expulsion (PBE). When PBE was observed, the oocytes were considered to mature at the MII stage.

The method has important significance for In Vitro Fertilization-Embryo Transfer (IVF-ET).

In a fourth aspect, the present invention claims the use of a substance capable of inhibiting the expression of a human OOSP2 protein and/or reducing the activity of a human OOSP2 protein in any one of:

p1, inhibiting the development of ova;

p2, preparing a product for inhibiting the development of ova;

p3, inhibiting oocyte maturation;

p4, preparing a product for inhibiting oocyte maturation.

Wherein said inhibiting development of an ovum is inhibiting development of an ovum in vitro; the inhibiting oocyte maturation is in vitro inhibiting oocyte maturation.

In a fifth aspect, the present invention claims the use of a substance capable of inhibiting the expression of a human OOSP2 protein and/or reducing the activity of a human OOSP2 protein in any one of:

q1, reducing the oocyte maturation rate;

q2, preparing a product for reducing the oocyte maturation rate;

q3, reducing the maturation rate of the oocyte;

q4, preparing a product for reducing the maturation rate of the oocyte.

Wherein, the reduction of oocyte maturation rate is a reduction of oocyte maturation rate in vitro; the reducing the oocyte maturation rate is reducing the oocyte maturation rate in vitro.

In the above fourth and fifth aspects, the substance capable of inhibiting the expression of human OOSP2 protein and/or reducing the activity of human OOSP2 protein may specifically be an antibody against the human OOSP2 protein.

In a sixth aspect, the invention claims a method for inhibiting oocyte maturation in vitro.

The method for inhibiting oocyte maturation in vitro as claimed in the present invention may include the steps of: inhibiting oocyte maturation is achieved by culturing immature oocytes (GV) in a medium containing sufficient antibodies against said human OOSP2 protein.

In a particular embodiment of the invention, the medium is an IVF medium. The medium is covered with a layer of mineral oil. In the method, each oocyte is cultured separately in a separate chamber in an embryoside dish (vitrolite). The culture conditions were 37 ℃,6% CO ₂ . The culture time is 72h.

In addition, the methods can be used to reduce oocyte maturation (rate) rate in vitro, and to inhibit egg development in vitro.

The method can be used for preparing corresponding disease models for drug screening.

In the above aspects, the human OOSP2 protein may be any one of:

(A1) Protein with amino acid sequence shown as SEQ ID No. 1;

(A2) Protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID No.1 and has the same function;

(A3) A protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity to the amino acid sequence defined in any one of (A1) to (A2) and having the same function;

(A4) A fusion protein obtained by attaching a protein tag to the N-terminus and/or C-terminus of a protein defined in any one of (A1) to (A3).

In the above proteins, the protein-tag refers to a polypeptide or protein that is expressed by fusion with a target protein using in vitro DNA recombination technology, so as to facilitate expression, detection, tracing and/or purification of the target protein. The protein tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and/or a SUMO tag, etc.

In the above proteins, identity refers to the identity of amino acid sequences. The identity of the amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web pages of the NCBI home website. For example, in the advanced BLAST2.1, by using blastp as a program, setting the value of Expect to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, per residual Gap cost, and Lambda ratio to 11,1 and 0.85 (default values), respectively, and performing a calculation by searching for the identity of a pair of amino acid sequences, a value (%) of the identity can be obtained.

In the above aspects, the nucleic acid molecule capable of expressing the human OOSP2 protein may be any one of the following DNA molecules:

(B1) DNA molecule shown in SEQ ID No. 2;

(B2) A DNA molecule which is hybridized with the DNA molecule defined in (B1) under strict conditions and codes the human OOSP2 protein;

(B3) A DNA molecule which has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% of identity with the DNA sequence limited by (B1) or (B2) and encodes the human OOSP2 protein.

In the above nucleic acid molecules, identity refers to the identity of nucleotide sequences. The identity of the nucleotide sequences can be determined using homology search sites on the Internet, such as the BLAST web page of the NCBI home website. For example, in the advanced BLAST2.1, by using blastp as a program, the Expect value is set to 10, all filters are set to OFF, BLOSUM62 is used as a Matrix, the Gap existence cost, the Per residual Gap cost and the Lambda ratio are set to 11,1 and 0.85 (default values), respectively, the identity of a pair of nucleotide sequences is searched, calculation is performed, and then the value (%) of identity can be obtained.

In the above nucleic acid molecule, the stringent conditions may be as follows: 50 ℃ in 7% Sodium Dodecyl Sulfate (SDS), 0.5M Na ₃ PO ₄ And 1mM EDTA, in 50 ℃,2 x SSC,0.1% SDS rinsing; also can be: 50 ℃ in 7% SDS, 0.5M Na ₃ PO ₄ And 1mM EDTA, and rinsing in 1 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M Na ₃ PO ₄ And 1mM EDTA, rinsed in 0.5 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M Na ₃ PO ₄ And 1mM EDTA, and rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; it can also be: 50 ℃ in 7% SDS, 0.5M Na ₃ PO ₄ Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 65 ℃; can also be: in 6 XSSC, 0.5%In a solution of SDS, hybridization was carried out at 65 ℃ and then the SDS and 1 XSSC, 0.1% by weight of SDS, each washing of the membrane was once performed with 2 XSSC, 0.1% SDS.

Experiments prove that the human OOSP2 protein can effectively induce oocyte maturation in vitro, and can improve the utilization rate of ova and improve pregnancy outcome in the test tube infant period. For patients with low egg maturation rates, such as patients with polycystic ovary syndrome, gonadotropin-stimulated high-response patients, ovarian low-response patients, etc., these patients cannot obtain enough mature eggs through the conventional ovulation promoting protocol. By utilizing human OOSP2 protein, the in vitro maturation rate of the ovum can be effectively improved, the utilization rate of the ovum of the patients can be improved, and the pregnancy outcome can be improved. Has important significance in IVF clinical application.

Drawings

FIG. 1 shows the recording of GVBD and PBE of ova using the Time-lapse real-Time microscopy system.

FIG. 2 shows the comparison of the maturation rate of eggs after adding human OOSP2 protein with that of a control group, which is 24 groups of eggs.

FIG. 3 shows the comparison of the maturation rate of ova after addition of OOSP2 antibody with that of the control group, for 16 groups of ova.

FIG. 4 is a record of maturation time of eggs from the same donor after addition of human OOSP2 protein. The immature state was recorded after 72h without expelling the polar body. The abscissa corresponds to different donors.

FIG. 5 is a record of maturation time of ova from the same donor after human OOSP2 antibody. The immature state was recorded after 72h without expelling the polar body. The abscissa corresponds to different donors.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, and the examples are given only for illustrating the present invention and not for limiting the scope of the present invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The amino acid sequence of the human OOSP2 protein related in the following examples is shown as SEQ ID No.1, and the coding gene thereof is shown as SEQ ID No. 2.

Example 1 human OOSP2 protein promotes maturation of ova

1. Experimental materials and methods

A total of 82 immature oocytes (GV) were collected from 36 donors (informed consent) (some donors donated more than 2 oocytes). To eliminate heterogeneity from different donors, each pair of oocytes from the same donor was divided into experimental and control groups.

To test the function of OOSP2 protein, the experimental group added hRec-OOSP2 (TP 308472, origene) which is a human-derived recombinant OOSP2 protein to IVF medium (G-IVF) at a final concentration of 150ng/mL ^TM PLUS, vivolife). The control group used IVF medium without addition.

Meanwhile, to test the inhibitory effect of the OOSP2 antibody, experimental groups added 1. Controls the same diluted concentration of IgG (12-370, millipore) was added to IVF medium.

Each oocyte was cultured in 25. Mu.l of medium covered with mineral oil and in a separate chamber in an Embryoslide Petri dish (vitrolite). 6% CO at 37 ℃ for each pair of oocytes ₂ Real-time photographic imaging was performed in a humid atmosphere using the embryoscopy time delay system (vitrolite) until 72 hours of incubation. Blastocyst Disruption (GVBD) and polar exclusion (PBE) are two phases of morphological changes after oocyte maturation under the microscope. Oocytes were considered to be matured at the MII stage when PBE was observed within 72 hours (FIG. 1), and were considered not to be matured if PBE was not observed within 72 hours.

2. Results and analysis of the experiments

For each set of experiments, paired GV stage oocytes from the same donor were compared. Of the 24 GV oocytes, 21 oocytes incubated with hRec-OOSP2 were matured (87.5%), whereas 16 oocytes matured in the control group (67%) (FIG. 2). Indicating a higher rate of maturation of the composition incubated with hRec-OOSP 2.

If OOSP2 functions as a secreted protein that induces oocyte maturation, addition of antibodies to OOSP2 during IVM may prevent or slow oocyte maturation. 10 of 16 GV oocytes incubated with OOSP2 antibody (ab-OOSP 2) were immature (62.5%); however, only 4 of the 16 GV oocytes in the control group were immature (25%) (fig. 3). This finding indicates that incubation of GV oocytes with antibodies against OOSP2 increases the rate of non-maturation.

Antibodies and recombination experimental results support the view of OOSP2 as an oocyte maturation factor.

In addition, the research of the invention also finds that: oocytes incubated with hRec-OOSP2 reached GVBD and PBE faster (approximately 2 hours) than control oocytes (FIG. 4). However, some oocytes incubated with ab-OOSP2 showed slower GVBD and PBE (approximately 5 hours) than control oocytes (FIG. 5).

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

<110> Qinghua university

Application of <120> OOSP2 protein in promotion of egg development

<130> CGGNQLN216129

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 158

<212> PRT

<213> Homo sapiens

<400> 1

Met Ala Leu Glu Val Leu Met Leu Leu Ala Val Leu Ile Trp Thr Gly

1 5 10 15

Ala Glu Asn Leu His Val Lys Ile Ser Cys Ser Leu Asp Trp Leu Met

20 25 30

Val Ser Val Ile Pro Val Ala Glu Ser Arg Asn Leu Tyr Ile Phe Ala

35 40 45

Asp Glu Leu His Leu Gly Met Gly Cys Pro Ala Asn Arg Ile His Thr

50 55 60

Tyr Val Tyr Glu Phe Ile Tyr Leu Val Arg Asp Cys Gly Ile Arg Thr

65 70 75 80

Arg Val Val Ser Glu Glu Thr Leu Leu Phe Gln Thr Glu Leu Tyr Phe

85 90 95

Thr Pro Arg Asn Ile Asp His Asp Pro Gln Glu Ile His Leu Glu Cys

100 105 110

Ser Thr Ser Arg Lys Ser Val Trp Leu Thr Pro Val Ser Thr Glu Asn

115 120 125

Glu Ile Lys Leu Asp Pro Ser Pro Phe Ile Ala Asp Phe Gln Thr Thr

130 135 140

Ala Glu Glu Leu Gly Leu Leu Ser Ser Ser Pro Asn Leu Leu

145 150 155

<210> 2

<211> 477

<212> DNA

<213> Homo sapiens

<400> 2

atggcgttag aagtcttgat gctcctcgct gtcttgattt ggaccggtgc tgagaacctc 60

catgtgaaaa taagttgctc tctggactgg ttgatggtct cagttatccc agttgcagaa 120

agcagaaatc tgtatatatt tgcggatgaa ttacatctgg gaatgggctg ccctgcaaat 180

cggatacata catatgtata tgagtttata tatcttgttc gtgattgtgg catcaggaca 240

agggtagttt ctgaggaaac tctccttttt caaaccgagc tgtactttac cccaaggaat 300

atagatcatg accctcagga aatccatttg gagtgttcca cctctaggaa atcagtgtgg 360

cttacaccag tttctactga gaatgaaata aaattggatc ctagtccttt tattgctgac 420

tttcagacaa cagcagaaga gttaggatta ttatcttcta gtccaaactt gctctga 477

Claims

1. Use of a human OOSP2 protein or a related biological material thereof in any of:

p1, promoting the development of ova;

p2, preparing a product for promoting the development of ova;

p3, inducing oocyte maturation;

p4, preparing a product for inducing oocyte maturation;

2. Use of a human OOSP2 protein or a related biological material thereof in any of:

q1, improving the oocyte maturation rate;

q2, preparing a product for improving the maturation rate of the oocyte;

q3, improving the maturation rate of the oocyte;

q4, preparing a product for improving the maturation rate of the oocyte;

3. A method for inducing oocyte maturation in vitro, comprising the steps of: the immature oocyte was cultured in a medium containing human OOSP2 protein at a final concentration of 150ng/mL, thereby obtaining a mature oocyte.

4. The application of substances capable of inhibiting the expression of human OOSP2 protein and/or reducing the activity of human OOSP2 protein in any one of the following substances:

p1, inhibiting the development of ova;

p2, preparing a product for inhibiting the development of ova;

p3, inhibiting oocyte maturation;

p4, preparing a product for inhibiting oocyte maturation.

5. The application of substances capable of inhibiting the expression of human OOSP2 protein and/or reducing the activity of human OOSP2 protein in any one of the following substances:

q1, reducing the oocyte maturation rate;

q2, preparing a product for reducing the oocyte maturation rate;

q3, reducing the maturation rate of the oocyte;

q4, preparing a product for reducing the maturation rate of the oocyte.

6. Use according to claim 4 or 5, characterized in that: the substance capable of inhibiting the expression of the human OOSP2 protein and/or reducing the activity of the human OOSP2 protein is an antibody against the human OOSP2 protein.

7. A method of inhibiting oocyte maturation in vitro comprising the steps of: and (2) culturing the immature oocyte in a culture medium containing a sufficient amount of antibodies against the human OOSP2 protein, thereby inhibiting maturation of the oocyte.

8. Use or method according to any of claims 1-7, wherein: the human OOSP2 protein is any one of the following proteins:

(A1) Protein with amino acid sequence shown as SEQ ID No. 1;

9. The use or method of any of claims 1-8, wherein: the nucleic acid molecule capable of expressing the human OOSP2 protein is any one of the following DNA molecules:

(B1) DNA molecule shown in SEQ ID No. 2;