CN113189334B

CN113189334B - Application of COMT protein in prediction of good response of crab eating monkey to superovulation

Info

Publication number: CN113189334B
Application number: CN202110460817.7A
Authority: CN
Inventors: 杨世华; 郭韵仪; 侯润杰
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2021-04-27
Filing date: 2021-04-27
Publication date: 2022-04-26
Anticipated expiration: 2041-04-27
Also published as: CN113189334A

Abstract

The invention relates to an application of COMT protein in predicting good response of crab eating monkey superovulation, belonging to the technical field of superovulation, wherein the biological information analysis is carried out on blood sample protein of the superovulation successful group on day 1, the superovulation successful group on day 5, the superovulation failed group on day 1 and the superovulation failed group on day 5 through a proteomics method to obtain differential protein among the groups. From the protein level studies, studies were conducted to reveal the changes in protein expression in the blood of superovulated monkeys. In the process of crab eating monkey superovulation, the COMT protein cannot be detected in the superovulation failure group, but still exists in the superovulation success group at day 5, possibly suggesting that the stabilization of the COMT protein expression level may be beneficial to superovulation. Therefore, the COMT protein becomes a new molecule for predicting good response of the super ovulation of the cynomolgus monkey, and has practical application value.

Description

Application of COMT protein in prediction of good response of crab eating monkey to superovulation

Technical Field

The invention relates to the technical field of superovulation, in particular to application of COMT protein in predicting good response of the superovulation of a cynomolgus monkey.

Background

Superovulation is a technique of stimulating the ovaries of female animals by using exogenous hormones to promote the simultaneous growth and development of multiple follicles in the ovaries, and finally obtaining multiple mature oocytes. Cynomolgus monkeys have only one dominant follicle in the natural menstrual cycle to mature and ovulate. If the superovulation technology is used in the menstrual period, the number of oocytes per ovulation can be increased from the original 1 to 10-20 or more. Mature oocytes are important materials for the development of human diseases, genetic engineering and embryo engineering studies. However, the number of oocytes that can be obtained in a natural cycle is very small, and thus the experimental requirements cannot be met, and the required cycle is too long. The superovulation can fully explore the reproductive potential of female animals, and can ensure that a large number of oocytes in ovaries grow, develop and mature. Therefore, obtaining a large number of mature oocytes by superovulation is an effective and feasible approach.

Although more mature oocytes than normal natural cycle are obtained in different superovulation schemes, there are large differences in the number of oocytes obtained from different animals. Relatively speaking, the superovulation effect of obese cynomolgus monkeys with the weight more than 5kg and the superovulation effect of cynomolgus monkeys with the weight less than 3kg are not ideal, and the number of mature oocytes in superovulation of the cynomolgus monkeys with the weight of 3-5 kg is large. In repeated superovulation, if the macaque has poor ovarian response during the first superovulation, the superovulation effect is not ideal during the subsequent superovulation. If the macaque with better ovarian response during the last superovulation is selected for repeated superovulation, the quantity and quality of the oocytes obtained in the next superovulation are more ideal. In addition, macaques with seasonal reproduction have significantly lower numbers of mature oocytes obtained by superovulation in the non-reproductive season than in the reproductive season. In addition, menstrual cycle disturbances, diarrhea and the presence of undegraded corpus luteum in the ovaries can also lead to undesirable effects of superovulation.

Disclosure of Invention

In order to overcome the problems in the prior art, the invention firstly provides the application of the COMT protein in predicting the good response of the super ovulation of the cynomolgus monkey.

The purpose of the invention is realized by the following technical scheme:

application of COMT protein in predicting good response of crab eating monkey superovulation.

The Catechol O Methyltransferase (COMT) gene is located at 22q11.21, spans 28.2kb, contains 6 exons, 5 introns, encodes Catechol O methyltransferase, and is expressed mainly in granulosa cells of the ovary, the stroma of the endometrium, and the gland. COMT is the major catabolic enzyme of estrogen metabolites, which inactivates the catechol estrogen by methylation, reducing its ability to bind to receptors. COMT breaks down the toxic products of estrogens such as 4-hydroxyestradiol (4-OH E2), 4-hydroxyestrone (4-OH E1) and 16 alpha-hydroxyestrone (16 alpha-OH E1) and converts them to methylation products which are soluble in water for elimination. An increase in estrogen metabolites may promote follicular atresia, leading to anovulation. The COMT protein is up-regulated during the menstrual cycle in the proliferative and early secretory stages and down-regulated during and after the secretory stage. COMT polymorphisms affect enzyme activity, cause a reduction in ovarian follicles, and are predictive of premature ovarian failure. Decreased COMT activity may increase the risk of hormone-dependent diseases by increasing estradiol levels in serum and tissues as well as accumulation of catechol estrogens and subsequent oxidative DNA damage.

Superovulation is always the key point of research on assisted reproductive technologies, and the number, maturity and development potential of oocytes obtained in superovulation are very important. The cynomolgus monkey can obtain ten or even dozens of mature MII-stage oocytes in normal superovulation, however, after repeated superovulation, part of the cynomolgus monkeys begin to respond in superovulation, can obtain only a small amount of GV-stage eggs, and perform poorly in subsequent superovulation. Although the ovary stores a large number of primordial follicles, it cannot be efficiently harvested by superovulation. Although numerous studies on superovulation have confirmed the presence of antibodies, body weight, season, age, etc. influencing factors in superovulation, superovulation is a relative regulation between gonadotropins and oocytes and is a complex dynamic process. Therefore, the mechanism affecting superovulation has not been elucidated and effective solutions have not been proposed. Since proteins are the final actors in life activities, the superovulation process is also the result of the relative action of a large number of proteins in the body.

In this experiment, a DIA non-standard quantitative technique was used, which divides the entire scan range of the mass spectrum into several windows and performs high-speed, cyclic selection, fragmentation and detection of all ions in each window. Therefore, compared with the traditional DDA technology, DIA can collect all fragment information in a sample, greatly improve data utilization rate, have few missing values, and have higher detection rate of low-abundance proteins. In the research, blood samples are grouped according to the superovulation result, and the blood samples subjected to superovulation of 6 cynomolgus monkeys are analyzed through a proteomics method to identify 1550 proteins in total, wherein the biological information analysis is performed on the sample proteins of the superovulation successful group on day 1, the superovulation successful group on day 5, the superovulation failed group on day 1 and the superovulation failed group on day 5, so as to obtain the difference proteins among the groups. From the protein level studies, studies were conducted to reveal the changes in blood protein expression during superovulation. Attempts to screen out related differential proteins influencing the quantity and quality of oocytes in superovulation provide possible molecular markers for further improving superovulation schemes and predicting superovulation results.

From the qualitative protein analysis, it was found that the COMT protein was present in menstrual blood samples, but on day 5 of the superovulation, both proteins were not detected in the response failure group and were still present in the response success group. In estrogen metabolism, the metabolite of COMT, 2-methoxyestradiol, has a potential physiological role in follicular homeostasis, normally at low levels during early follicular development and at higher levels in the fully developed dominant follicle. Modulation of COMT activity and 2-methoxyestradiol concentration is part of ovarian physiology. Disturbances in 2-methoxyestradiol levels may be associated with increased follicle depletion, eventually leading to follicular arrest. In patients with polycystic ovary syndrome, overexpression of catechol methyltransferase and elevated levels of 2-methoxyestradiol in ovarian granulosa cells results in abnormal steroidogenesis, follicular arrest and no ovulation. In patients with primary ovarian insufficiency, the opposite occurs, with an increased incidence of low activity of the COMT allele, promoting a reduction in the formation of 2-methoxyestradiol. Thus, pathological changes in COMT may lead to major disturbances in follicular formation. Similarly, in the superovulation failure group, the disappearance of COMT protein at day 5 may result in a disturbed level of 2-methoxyestradiol, affecting follicular formation and leading to failure of the superovulation response. In the superovulation response successful group, the stabilization of the COMT protein expression level may be beneficial to superovulation.

Preferably, the COMT protein is used as a positive regulator for predicting good response to superovulation in cynomolgus monkeys.

Thus, in particular predicting the success or failure of superovulation in cynomolgus monkeys, it can be predicted by detecting the presence or absence of the COMT protein in blood samples from day 1 to day 5 of the cynomolgus monkey superovulation.

Of course, when the cynomolgus monkey superovulates, the hormone may be administered according to the existing scheme, which includes but is not limited to the following three:

the first method comprises the following steps: female cynomolgus monkeys were subcutaneously injected with 3.75mg of GnRH agonist on day 1 of menstruation. Two to three weeks later, female cynomolgus monkeys injected 75IU/kg rhFSH subcutaneously, three times every 72 hours, and 60 hours after the last injection, 1200IU hCG was intravenously injected.

And the second method comprises the following steps: the female monkey was administered with 60IU of rhFSH twice daily for 6 consecutive days, 60IU of rhFSH twice daily for 3 consecutive days, and then 5 hours later with 1000IU of hCG intravenously. To suppress premature ovulation, 0.25mg of GnRH inhibitor was injected daily starting on day 1 of stimulation of rhFSH and continuing until the day of hCG.

And the third is that: on day 1 of menstruation, female cynomolgus monkeys were injected subcutaneously with 3.75mg of GnRH agonist. After two to three weeks, follicular growth was stimulated by either the a or B method. The method A comprises the following steps: 25IU/kg rhFSH was dissolved in glycerol/physiological saline (1:1) and injected subcutaneously once a day for 9 days, and after 36h, 1200IU hCG was injected intravenously. The method B comprises the following steps: female cynomolgus monkeys injected 75IU/kg hFSH subcutaneously every 3 days, and after 60h interval for the last treatment, 1200IU hCG intravenously.

Compared with the prior art, the invention has the following beneficial effects:

according to the invention, through a proteomics method, biological information analysis is carried out on the sample proteins of the superovulation successful group on day 1, the superovulation successful group on day 5, the superovulation failed group on day 1 and the superovulation failed group on day 5, so as to obtain the differential proteins among the groups. From the protein level studies, studies were conducted to reveal the changes in blood protein expression during superovulation. Attempts to screen out related differential proteins influencing the quantity and quality of oocytes in superovulation provide possible molecular markers for further improving superovulation schemes and predicting superovulation results.

In the process of crab eating monkey superovulation, the COMT protein cannot be detected in the superovulation failure group, but still exists in the superovulation success group at day 5, possibly suggesting that the stabilization of the COMT protein expression level may be beneficial to superovulation.

The prediction provides a new method for predicting the good response of the crab eating monkey to the superovulation, and has practical application value.

Drawings

FIG. 1 is a flow chart of the proteomics experimental procedures and results analysis of the present invention;

FIG. 2 shows the results of quantitative analysis of differential protein; wherein FIG. 2a is a histogram of the differential protein; FIG. 2b is a Wien diagram of differential proteins and total number of identified proteins.

Detailed Description

The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

The test methods used in the following experimental examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

One, superovulation experimental process

Physiological status of adult female cynomolgus monkeys in the target monkey cohort was observed daily and cage and monkey number registration was made on day 1 where menses were found to occur. On days 1-4 of menstruation, venous blood sample collection, B-type ultrasonic image examination of ovary and uterus and color ultrasonic image examination of ovary and uterus are carried out on cynomolgus monkeys in menstrual period. Meanwhile, the intramuscular injection of rhFSH was started on the evening of the day the menstrual blood sample was collected, and the day was recorded as day 1 of the superovulation. rhFSH was injected once a day, in the morning and in the evening, starting on the second day. On day 5 of injection of rhFSH, blood sample collection was again performed. The needle was stopped after completion of the intramuscular injection of rhFSH on the 9 th morning of superovulation. The superovulation cynomolgus monkeys were given an intramuscular injection of hCG once on the evening of day 10 of superovulation. Finally, blood sample collection and laparoscopic ovum retrieval were performed 36 hours after rhCG injection and on the 12 morning of superovulation.

Proteomics detection and result analysis

Screening 3 cynomolgus monkeys with poor and good superovulation responses, placing 2mL blood samples collected on day 1 and day 5 in a precooled 4 ℃ centrifuge, centrifuging at 3000rpm for 10 minutes, taking supernatant in an EP tube, and storing in a refrigerator at-80 ℃.

The blood samples were divided into 4 groups, which were day 1 in the super-exclusion failure group, day 5 in the super-exclusion failure group, day 1 in the super-exclusion success group, and day 5 in the super-exclusion success group, respectively. The 4 groups of plasma samples were subjected to subsequent work of proteomics, and the 4 groups of proteins were analyzed in comparison with each other until protein data was generated. The proteomics method selects DIA non-labeled quantitative proteomics technology to complete analysis. Under the data independent acquisition mode, a large amount of proteome coverage can be provided, and simultaneously, a large amount of proteins in each sample can be accurately and highly repeatable quantified, so that the quantitative analysis platform is an ideal proteome quantitative platform for differential proteome qualitative analysis or mass samples. The specific experimental process and the information analysis process are shown in fig. 1. The protein sequences finally identified in proteomics were all derived from the UniProt protein database, NCBI and Ensembl gene annotation system, and were done by the Spectronaut software, with the differential analysis done by the RStudio software.

The data results of four groups of blood sample proteomics are screened and integrated, and the study is carried out from protein characterization and differential analysis.

The experimental results are as follows: blood proteins of female cynomolgus monkeys with poor superovulation response on day 1 and day 5 were designated as Treated1 group (T1) and Treated 2 group (T2), respectively, and blood proteins of female cynomolgus monkeys with good superovulation response on day 1 and day 5 were designated as Treated 3 group (T3) and Treated 4 group (T4), respectively. The first part is to compare and analyze the differential protein between the super-exclusion failure group T1 and T2 and the differential protein between the super-exclusion success groups T3 and T4, and compare the change of the differential protein in the super-exclusion process between the super-exclusion failure group and the super-exclusion success group; the second part is to analyze the differential protein of the superovulation failure group T1 and the superovulation success group T3 and compare the difference of the blood protein of the superovulation failure group and the superovulation success group in the menstrual period; and in the third part, the differential proteins of the superovulation failure group T2 and the superovulation success group T4 are analyzed, and the differential proteins of blood in the superovulation process between the superovulation success group and the superovulation failure group are compared. Wherein each portion of the differential protein is to be analyzed in terms of Gene Ontology (GO), pathway enrichment analysis (KEGG), interactions, etc. of the differential protein.

As shown in fig. 2a, the quantitative analysis of differential proteins revealed that T1 had a total of 56 differential proteins compared to T2, of which 52 were up-regulated and 4 were down-regulated.

Since the differential protein analysis results were performed on the differential levels of proteins present in all 4 groups of samples, 55 proteins were not detected in some of the 4 groups of samples, as shown in fig. 2b, in addition to 146 differential proteins among the identified proteins, including 60S ribosomal protein L26(60S ribosomal protein L26, RPL26), 40S ribosomal protein S9(40S ribosomal protein S9, RPS9), Catechol O-methyltransferase (COMT). Among them, RPL26 protein was not detected on day 5 of the superovulation group, but was present in all other groups. In the GO assay, COMT proteins are mainly involved in biological processes such as methylation, neurotransmitter breakdown, and the like. In the KEGG analysis, COMT proteins are mainly involved in signal pathways such as steroid hormone biosynthesis, tyrosine metabolism, and the like.

Claims

1. The application of the blood COMT protein in predicting the good response of the super-ovulation of the cynomolgus monkey, wherein the COMT protein is used as a positive regulatory factor for predicting the good response of the super-ovulation of the cynomolgus monkey.