CN108344868B

CN108344868B - Application of human histones H3Ser10 and Ser28 in identification of development stage of human early embryo

Info

Publication number: CN108344868B
Application number: CN201810064065.0A
Authority: CN
Inventors: 陈磊; 马宁赵
Original assignee: First Affiliated Hospital of Zhengzhou University
Current assignee: First Affiliated Hospital of Zhengzhou University
Priority date: 2018-01-23
Filing date: 2018-01-23
Publication date: 2020-05-15
Anticipated expiration: 2038-01-23
Also published as: CN108344868A

Abstract

The invention relates to a new application of a product for detecting phosphorylation states of human H3Ser10 and H3Ser 28. The phosphorylation state of H3Ser10 can be used for identifying or assisting in identifying the number of male pronuclei in human embryos, the development period of human embryos, the growth period of human oocytes, or screening human embryos or human ova; the phosphorylation state of H3Ser28 can be used to identify or aid in identifying the stage of human embryo development, the stage of growth of human oocytes, or screening for human embryos or human ova.

Description

Application of human histones H3Ser10 and Ser28 in identification of development stage of human early embryo

Technical Field

The invention relates to the technical field of human assisted reproduction, in particular to application of human histone H3Ser10 (serine at position 10) and Ser28 (serine at position 28) in identifying the development stage of a human early embryo.

Background

Epigenetics is a branch of genetics that is the study of changes in gene expression that can be transmitted to the next generation, with the proviso that changes in non-DNA sequences are not being made. Its research categories include DNA methylation (DNA methylation), histone post-translational modifications (PTMs), genomic imprinting (genomic typing), RNA editing (RNA editing), gene silencing (gene silencing), maternal effects (matrix effects) and activation of dormant transposons. Epigenetics is not only involved in maintaining normal proliferation and apoptosis of cells, but also closely related to embryonic development and malignant transformation of cells. Precise and complex epigenetic modification changes are required at each step in the germ cell growth and in the post-fertilization embryonic development.

Among genetic epigenetic modifications, histone post-translational modification is a more common modification. It includes histone phosphorylation, acetylation, methylation, ADP ribosylation, ubiquitination, etc. Wherein, histone phosphorylation is the phosphorylation modification process of histone N-terminal amino acid residue, and the main sites of phosphorylation of histone H3 are serine at position 10 (Ser10), serine at position 28 (Ser28), threonine at position 3 (Thr3) and threonine at position 11 (Thr 11). Among the mammalian histone phosphates, the most representative ones are the phosphorylation of histone H3Ser10 (H3 serine at position 10) and H3Ser28 (serine at position 28), and the distribution and expression of H3Ser10 phosphorylation and H3Ser28 phosphorylation changes as the oocyte matures through meiosis.

The studies of wangqiang et al on H3Ser10 and H3Ser28 in mice showed that in oocytes in the Germinal Vesicle (GV) stage, H3Ser10 phosphorylated had a significant signal and co-localized with DNA, and after resumption of meiosis, the signal phosphorylated by H3Ser10 was spread throughout the chromosome, but was the strongest in heterochromatin surrounding the centromere in the middle of the first meiosis; however, Swain et al showed that phosphorylated H3Ser10 was not found in the mouse GV phase. In the research on the maturation process of the porcine oocytes, the phosphorylation signal of H3Ser10 in the GV stage is relatively weak, the phosphorylation signal of H3Ser10 is gradually enhanced along with the rupture of the blastocyst, and the phosphorylation signal is distributed on the whole chromosome from the metaphase to the metaphase of the second division; the H3Ser28 phosphorylation signal is weak at the GV stage, and it surrounds the chromosome from Pre-MI to MII, and disappears at a later stage. Studies on histone phosphorylation in rabbit oocytes showed that H3Ser10 phosphorylation occurred during the blastocyst disruption phase and reached a peak during the blastocyst disruption phase, partial dephosphorylation occurred during the first meiosis, the phosphorylation signal was weak, but the phosphorylation level slowly increased again during the second meiosis.

As can be seen from the existing report, the phosphorylation of histone H3 is representative in the oocyte maturation process, but the phosphorylation pattern is different among different species, especially different research results are provided for the same species (mouse), and particularly, the phosphorylation state of histone H3 in the human oocyte maturation process is not reported at present.

The invention adopts specific antibody and immunofluorescence technology, explores histone H3 phosphorylation process in human oocyte maturation process and early embryo development process, and fills in the gap of basic research related to human assisted reproductive technology.

Disclosure of Invention

As used herein, the term "human histone H3Ser 10" refers to serine at position 10 of human histone H3, and "Ser 28" refers to serine at position 28, and as used herein, unless otherwise specified, H3Ser10 refers to serine at position 10 of human histone H3, and H3Ser28 refers to serine at position 28 of human histone H3.

In one aspect, the invention provides a product for detecting phosphorylation of human histones H3Ser10 and Ser28, wherein the product is preferably an antibody, preferably a monoclonal antibody, against phosphorylation of human histones H3Ser10 and Ser 28.

In another aspect, the invention provides the use of a product for detecting phosphorylation of human histones H3Ser10 and Ser28 to identify human oocyte maturation stages including the GV stage (germinal vesicle stage), the MI stage (metaphase first meiosis), the ATI stage (telase after first meiosis) and the MII stage (metaphase second meiosis).

In one embodiment, phosphorylation of H3Ser10 is present in the GV, MI, ATI and mil phases of human oocytes, phosphorylation of H3Se28 is present in the MI, ATI and mil phases of human oocytes, and phosphorylation of H3Ser28 is not present in the GV phase of human oocytes.

In one embodiment, the simultaneous phosphorylation of H3Ser10 and H3Ser28 indicates that the human oocyte is in MI phase, ATI phase and MII phase, the phosphorylation of H3Ser10, and the non-phosphorylation of H3Ser28 indicates that the human oocyte is in GV phase, and the detection of the phosphorylation states of H3Ser10 and H3Ser28 can improve the accuracy of the identification result.

Preferably, the oocyte is derived from ICSI (intracytoplasmic sperm injection)

In another embodiment, the products of detecting phosphorylation of human histones H3Ser10 and Ser28 can also be used to identify the developmental stage of human early embryos including the prokaryotic stage where genetic material of the ovum and sperm forms two clearly visible pronuclei after the sperm enters the ovum and the 1-cell stage where the embryo is at the stage before the first cell division after the disappearance of the pronuclei.

In one embodiment, H3Ser10 phosphorylation occurs predominantly in the male pronuclei during the prokaryotic phase of human early embryos and is evenly distributed on the chromosome at the 1-cell phase; h3Ser28 did not show phosphorylation signals in the prokaryotic phase of human early embryos, but as the embryos further developed, it was seen to be located on chromosomes at 1-cell phase.

In one embodiment, simultaneous phosphorylation of H3Ser10 and H3Ser28 indicates that the human early embryo is in the 1-cell stage, and phosphorylation of H3Ser10, but not phosphorylation of H3Ser28 indicates that the human early embryo is in the prokaryotic stage.

Preferably, the human early embryo is derived from IVF (in vitro fertilization) and/or ICSI (intracytoplasmic sperm injection).

Preferably, the human early embryo is a 2 prokaryotic (2 PN) embryo or a 3 prokaryotic (3 PN) embryo.

In another embodiment, phosphorylation by H3Ser10, but not H3Ser28, indicates that the human early embryo is in the prokaryotic stage, while the number of pronuclei phosphorylated by H3Ser10 may reflect the number of male pronuclei; when the H3Ser10 of n pronuclei in the human early embryo at the pronucleus stage is phosphorylated, the number of the male pronuclei in the human early embryo is judged to be n, preferably, n is an integer, and n is more than or equal to 1.

In another aspect, the present invention also provides a kit for identifying a human oocyte maturation stage, the kit including a product for detecting phosphorylation of human histones H3Ser10 and Ser28, the human oocyte maturation stage including a GV stage (blastocyst stage), an MI stage (metaphase of first meiosis), an ATI stage (late postmeiosis of first) and an MII stage (metaphase of second meiosis).

On the other hand, the invention also provides a kit for identifying the development stage of the human early embryo, which comprises a product for detecting phosphorylation of human histone H3Ser10 and Ser28, wherein the development stage of the human early embryo comprises a prokaryotic stage and a 1-cell stage.

In one embodiment, the product for detecting phosphorylation of human histones H3Ser10 and Ser28 is an antibody, preferably a monoclonal antibody, against phosphorylation of human histones H3Ser10 and Ser28, the kit comprises a primary antibody against H3Ser10 phosphorylation monoclonal antibody and a primary antibody against H3Ser28 phosphorylation monoclonal antibody, and preferably, the kit further comprises the reagent, a monoclonal antibody against endoglin (such as α -tubulin) and other reagents related to immunofluorescence staining detection, such as a labeled secondary antibody.

In another aspect, the present invention also provides a method of identifying a human oocyte maturation stage comprising the step of detecting phosphorylation of human histones H3Ser10 and H3Ser28, said human oocyte maturation stage comprising a GV stage (blastocyst stage), an MI stage (metaphase first meiosis), an ATI stage (telophase first meiosis) and an MII stage (metaphase second meiosis). In one embodiment, the simultaneous phosphorylation of H3Ser10 and H3Ser28 indicates that the human oocyte is in the MI phase, ATI phase and MII phase, and in another embodiment, the phosphorylation of H3Ser10 and the non-phosphorylation of H3Ser28 indicates that the human oocyte is in the GV phase. Preferably, the detection of phosphorylation of human histones H3Ser10 and H3Ser28 is performed by immunoassay, more preferably, by monoclonal antibody.

In another aspect, the invention also provides a method of identifying developmental stages of human early embryos comprising the step of detecting phosphorylation of human histones H3Ser10 and H3Ser28, including a prokaryotic stage and a 1-cell stage. In one embodiment, simultaneous phosphorylation of H3Ser10 and H3Ser28 indicates that the human early embryo is in the 1-cell stage, and in another embodiment, phosphorylation of H3Ser10, but not H3Ser28 indicates that the human early embryo is in the prokaryotic stage, preferably, the phosphorylation of human histones H3Ser10 and H3Ser28 is detected by immunoassay, more preferably, using a monoclonal antibody, preferably, the human early embryo is a 2 prokaryotic (2 PN) embryo or a 3 prokaryotic (3 PN) embryo.

On the other hand, the invention also provides a method for identifying the number of male pronuclei in the human early embryo, which comprises the step of detecting the phosphorylation of human histones H3Ser10 and H3Ser28, specifically, the phosphorylation of H3Ser10 and the non-phosphorylation of H3Ser28 indicate that the human early embryo is in the pronucleus stage, and at the moment, the number of pronuclei phosphorylated by H3Ser10 can reflect the number of male pronuclei; when the H3Ser10 of n pronuclei in the human early embryo at the pronucleus stage is phosphorylated, the number of the male pronuclei in the human early embryo is judged to be n, preferably, n is an integer, and n is more than or equal to 1.

The invention has the following beneficial effects:

the phosphorylation states of H3Ser10 and H3Ser28 are detected in the maturation process of the human oocyte and the development process of the human early embryo for the first time, and the accuracy of identification on the maturation stage and the early embryo development stage of the human oocyte can be improved by jointly detecting the phosphorylation states of H3Ser10 and H3Ser 28.

Drawings

FIG. 1 shows immunofluorescence staining patterns of human oocytes at GV, MI, ATI and MII stages, secondary antibody against H3ser10ph antibody is FITC conjugated (red), Hoechest is used to stain DNA in cells (blue), α -tubulin (green), and the slide is observed under a confocal laser microscope with a scale of 10 μm.

FIG. 2 is an immunofluorescent staining pattern of human oocytes at GV, MI, ATI and MII stages, with FITC-conjugated secondary antibody against H3ser28ph (green), Hoechest used to stain DNA in cells (blue), α -tubulin (red), slide 10 μm in scale, as viewed under a confocal laser microscope.

FIG. 3 shows immunofluorescence staining of human 3PN zygotes, with FITC-conjugated secondary antibody against phosphorylated antibody of H3ser10 (red), Hoechest for staining DNA in cells (blue), α -tubulin (green), FITC-conjugated secondary antibody against phosphorylated antibody of H3ser28 (green), Hoechest for staining DNA in cells (blue), α -tubulin (red), p for male pronuclei, m for female pronuclei, and slide 10 μm in scale, as observed under confocal laser microscopy.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 materials and methods

1. Study object

The study was targeted to patients undergoing assisted pregnancy therapy with test tube infants at the reproductive center of the first subsidiary hospital of zhengzhou university. Inclusion criteria were: 1. the age is less than or equal to 35 years old; 2. follicle Stimulating Hormone (FSH) < 12 mIU/L; 3. the volitional scheme is a long scheme or an overlong scheme. Patients had signed an informed consent, which was approved by the medical ethics committee of zheng zhou university.

2. Retrieval of oocytes

The oocytes collected were oocytes that were immature at the time of intracytoplasmic sperm microinjection (ICSI). Conventionally, ova were collected 36h after injection of human chorionic gonadotropin (Hcg), and intracytoplasmic single sperm microinjection was performed 4-6 h after the ova collection. At this time, only oocytes that have grown to the metaphase of the second meiosis are considered to be mature oocytes and can be injected. We collected the remaining immature oocytes in the blastocyst stage or first meiotic stage, either directly fixed or continued in vitro culture.

3. Ovulation induction protocol and egg retrieval procedure

The long scheme is as follows: in the middle of the last menstrual period, the drug is downregulated and completely downregulated by a gonadotropin releasing hormone agonist (GnRH-a, dabiejia, huilin, Switzerland, or Dafeline, Yipusheng, France) (i.e., serum FSH < 5mIU/mL, E2 < 50pg/mL LH < 5mIU/mL, endometrial thickness < 5mm, maximum follicle diameter < 5mm in bilateral ovaries). Ovulation induction by using genetic recombination follicle growth-promoting hormone (r-FSH, Gunafen, Merchano, Switzerland, or Prikang, Darmandon, USA or Ford Mongolian, Ebosa, Switzerland) or urogenic follicle stimulating hormone (HMG, Lizhu, China, or Helmetz, Huiling, Switzerland) on days 3-5 of the month, and adjusting the amount of Gn according to the size and endocrine level of the follicle, wherein the number of follicles of the largest follicle is more than 20mm or more than 16mm accounts for more than 2/3 of the total number, and using HCG trigger. Ova were collected by vaginal Ovarian Puncture (OPU) 36h after hCG injection, guided by ultrasound B.

The overlong scheme comprises the following steps: subcutaneously injecting long-acting gonadotropin releasing hormone agonist 3.75mg (Daphne, Yipu Sheng, France) at 2-3 days of menstruation at the initial stage of menstruation, and performing vaginal B ultrasonic examination after 28 days; ovulation promotion is performed according to the period of a patient, the dosage of Gn is adjusted according to the size of follicles and the endocrine level, and HCG trigger is used when the number of the follicles with the maximum size of more than 20mm or more than 16mm accounts for more than total number 2/3. After 36h of hCG injection, the ovum is taken out by vaginal ovary puncture under the guidance of B-ultrasonic.

Vaginal irrigation is performed 1 day before egg taking operation, vaginal irrigation is performed again on the same day of operation, and a towel is placed. Intravenous anesthesia is also used. Before the puncture needle enters, the position of a pelvic cavity blood vessel is firstly confirmed by ultrasound, and then the position and the size of bilateral ovaries, the number and the size of follicles and the condition of endometrium are determined. The puncture frame is arranged, the needle is inserted from the fornix behind the vagina or the fornix on the side, all the follicles are punctured one by one from near to far along the puncture line under the guidance of ultrasonic monitoring, and the aspiration negative pressure is 15 kpa. The follicular fluid of mature follicles is mostly pale yellow, and the final part is bloody. Puncturing follicles with the diameter of more than 10mm one by one; and after one side puncture is finished, the opposite side puncture is switched.

The obtained egg crown dune compound is quickly sent to a laboratory, an egg picker in the laboratory firstly observes whether a semitransparent amorphous loose mucus block exists or not by naked eyes, then observes and confirms a Cumulus Oocyte Complex (COC) under a dissecting mirror, and moves the COC into a G-MOPS dish by a Pasteur pipette. If blood clots or coarse and dark granular cells and the like exist around the COC complex, the COC complex can be cut off by a pipette under a dissecting mirror, and then the COC is placed into an egg picking dish. Removing blood cells by pipetting COC into Falcon3001 dish (60ml G-IVFTM with 6.7ml SSSTM) with another pipette, placing COC into culture solution, placing at 37 deg.C and 6% CO₂In an incubator. 4. Obtaining waste embryos

The human early embryo is discarded embryo which does not meet the transplantation and freezing standards In Vitro Fertilization (IVF) -embryo transplantation assisted pregnancy patients, the embryo source comprises ① cleavage stage development delay, 2 prokaryotic (2 PN) embryo which is not suitable for transplantation and freezing standards according to the III-IV grades evaluated by the conventional fixed point morphological system, ② abnormal fertilization 3 prokaryotic (3 PN) embryo, the source of the blastula comprises 1, the third day is obtained by discarded embryo culture which can not be transplanted or frozen, and 2, the discarded blastula which does not meet the transplantation standards is obtained by the patients cultured by the blastula (the inner cell mass and the trophoblast are graded as C grade).

5. Primary antibodies and reagents

6. Culture of waste oocytes

Upon collection of intracytoplasmic sperm microinjection, immature oocytes are transferred in groupsThe overnight equilibrated droplets of mineral oil-coated follicular medium were incubated at 37 ℃ with 6% CO₂The incubator (2) is observed regularly.

7. Culture of waste embryos

Collecting waste embryos that could not be transplanted and frozen on the third day of the center, transferring into mineral oil-covered blastocyst culture medium droplets that had been equilibrated overnight according to embryo source and experimental groups, placing in 37 deg.C, 5% CO₂Continuously culturing in the incubator until the blastocyst is reached, and observing and changing the culture solution every day.

8. Human oocyte and embryo immunofluorescence staining experimental procedure

1) Oocytes or embryos collected from the culture chamber were rapidly placed in 4% paraformaldehyde fixing solution and fixed for 30 min. 2) The oocytes or embryos are transferred from the fixative to a 0.5% Triton-X-100 permeabilizing solution for 20 min. 3) Transferring the oocyte or embryo into a sealing solution containing 1% of natural bovine serum albumin, and sealing for 1 h. 4) The primary antibody was diluted 1:100 with blocking solution to make 40. mu.l droplets, and cells were added to the droplets and incubated overnight in a refrigerator at 4 ℃. 5) The next day, the oocytes or embryos were washed three times for 5min each time with an eluent containing 1% Tween-20 and 0.01% Trition-X-100. 6) The secondary antibody was diluted 1:200 with the eluent to make 40. mu.l droplets, and the cells were transferred to the droplets and incubated for 1 h. Care was taken to avoid light. 7) After the secondary antibody incubation was completed, the eluent was eluted again three times for 5min each time. 8) Staining was performed with Hoechest (sigma,1ug/ml) for 15min at room temperature. 9) A40 ul drop of anti-quencher was dropped in the middle of the glass slide and the cells were placed in this drop. 10) Coverslips were mounted and viewed under a confocal laser microscope (Zeiss LSM 710).

9. Statistical treatment

The above experiments were performed in at least three independent replicates of oocytes or early embryos at each stage.

Example 2 localization of H3Ser10 and H3Ser28 phosphorylation in human oocytes

104 oocytes were collected in total, 28 GV, 26 MI, 24 ATI and 26 MII, which were divided into groups H3Ser10 and H3Ser28, and immunofluorescence staining analysis was performed on the 10 th serine phosphorylated monoclonal antibody of anti-histone H3 and the 28 th serine phosphorylated monoclonal antibody of anti-histone H3, respectively.

As shown in FIG. 1, the results of immunofluorescence analysis of the 10 th serine phosphorylation monoclonal antibody against histone H3 show that H3Ser10 phosphorylation is always present during oocyte maturation (see column H3/Ser10-P in FIG. 1), and starts in the GV stage oocyte, and is co-localized with DNA and distributed on chromosomes.

As shown in FIG. 2, the immunofluorescence analysis result of 28 th serine phosphorylation monoclonal antibody of anti-histone H3 shows that no phosphorylation signal of H3Ser28 is found in the GV stage during oocyte maturation, and the phosphorylation signal of H3Ser28 can be observed in MI stage along with the resumption of meiosis and continues to the second meiosis metaphase (MII stage).

Example 3 localization of H3Ser10 and H3Ser28 phosphorylation in IVF-derived 3PN early embryos

85 PN zygotes were collected and equally divided into groups H3Ser10 and H3Ser28, and immunofluorescence staining analysis of the 10 th serine phosphorylated monoclonal antibody against histone H3 and the 28 th serine phosphorylated monoclonal antibody against histone H3 was performed, respectively.

The results are shown in FIG. 3: immunofluorescence staining shows that in human 3PN fertilized eggs, the phosphorylation positions of H3Ser10 and H3Ser28 are different, and H3Ser10 phosphorylation mainly occurs in a male pronucleus in a 3PN pronucleus stage, and is uniformly distributed on a chromosome in a 1-cell stage along with further development. Whereas no phosphorylation signal was observed for H3Ser28 at the 3PN prokaryotic stage, but it was seen to be located chromosomally at 1-cell stage as the embryo was further developed.

The present invention is further illustrated in detail by the above embodiments in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the above-described specific embodiments are merely illustrative of the present invention and are not intended to limit the scope of the present invention.

Claims

1. Detecting the use of products phosphorylated by human histones H3Ser10 and H3Ser28 in identifying developmental stages of human early embryos, including the prokaryotic stage and the 1-cell stage;

the method is characterized in that the simultaneous phosphorylation of H3Ser10 and H3Ser28 indicates that the human early embryo is in a 1-cell stage;

phosphorylation of H3Ser10 and non-phosphorylation of H3Ser28 indicate that the human early embryo is in prokaryotic stage.

2. The use according to claim 1, characterized in that the products for detecting phosphorylation of human histones H3Ser10 and H3Ser28 are selected from antibodies directed against phosphorylation of human histones H3Ser10 and H3Ser 28.

3. Use according to claim 2, characterized in that the antibody is a monoclonal antibody.

4. Use according to any one of claims 1 to 3, wherein the human early embryo is a 2 or 3 prokaryotic (2 PN) embryo.

5. Use according to claim 4, wherein the human early embryo is derived from IVF (invitro fertilization).

6. The application of products phosphorylated by human histones H3Ser10 and H3Ser28 in identifying the number of male pronuclei in a human early embryo is detected, and is characterized in that the developmental stages of the human early embryo comprise a pronucleus stage and a 1-cell stage; when H3Ser10 is phosphorylated and H3Ser28 is not phosphorylated, the number of pronuclei phosphorylated by H3Ser10 is the number of male pronuclei in the early embryo of the human.

7. A method for identifying the development stage of a human early embryo is characterized by comprising the step of detecting phosphorylation of human histones H3Ser10 and H3Ser28, wherein the development stage of the human early embryo comprises a prokaryotic stage and a 1-cell stage, and the method is characterized in that simultaneous phosphorylation of H3Ser10 and H3Ser28 indicates that the human early embryo is in the 1-cell stage, and phosphorylation of H3Ser10 and non-phosphorylation of H3Ser28 indicate that the human early embryo is in the prokaryotic stage.

8. The method of claim 7, wherein the detecting phosphorylation of human histones H3Ser10 and H3Ser28 is by immunoassay.

9. The method of claim 8, wherein the immunoassay is performed using a monoclonal antibody.

10. The method of any one of claims 7-9, wherein the human early embryo is a 2 prokaryotic (2 PN) embryo or a 3 prokaryotic (3 PN) embryo.

11. The method of claim 10, wherein the human early embryo is derived from IVF (in vitro fertilization).

12. A method for identifying the number of male pronuclei in human early embryos, comprising the steps of detecting phosphorylation of human histones H3Ser10 and H3Ser28, wherein the developmental stages of the human early embryos comprise a pronuclei stage and a 1-cell stage; when H3Ser10 is phosphorylated and H3Ser28 is not phosphorylated, the number of pronuclei phosphorylated by H3Ser10 is the number of male pronuclei in the early embryo of the human.