Construction method and application of gene editing mouse model for researching calcium library function and low fertility in mature sperms

Abstract

The invention relates to the technical field of animal model construction, in particular to a construction method and application of a gene editing mouse model for studying calcium library function and low fertility in mature sperms.The model is characterized in that tamoxifen is used for inducing, a Ddx4ERT2 promoter is used for driving Cre recombinase expression, three subtypes of IP3R are specifically knocked out in sperms at the same time, and the mouse model is obtained. The method is used for researching the action and mechanism of the subtype in male fertility. After the model mouse is subjected to two rounds of induced knockout, the fertility is severely damaged, the sperm quantity and movement level are reduced, and the acrosome reaction is abnormal, so that the model is a good model for evaluating the sperm dysfunction, and is expected to be used for screening treatment drugs for improving the sperm dysfunction, especially male sterility diseases caused by advanced acrosome reaction in the future. In addition, through screening optimization of an IP3R agonist and a calcium signal detection platform, it is further confirmed that the mouse model is a good model for studying sperm calcium library mediated calcium signals.

Description

Technical Field

[0001] This invention relates to the field of animal model construction technology, and in particular to a method for constructing a gene-edited mouse model for studying the calcium store function in mature sperm and low fertility, and its application. Background Technology

[0002] Reproductive health issues have become an increasingly prominent social concern in modern society, with approximately one in six couples facing fertility problems. Male factors account for about half of these cases. The main male factors stem from abnormal spermatogenesis and sperm dysfunction. While numerous animal models exist for studying abnormal spermatogenesis, research on sperm dysfunction has been significantly diminished due to the widespread use of assisted reproductive technologies (ART). ART, including in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), can resolve more than half of infertility cases caused by sperm dysfunction. However, ART violates the natural laws of conception, and mounting evidence suggests that offspring conceived through ART carry various health risks, such as a significantly increased risk of neurological disorders and cancer. Therefore, researching the causes and mechanisms of mature sperm dysfunction, enabling these patients to conceive naturally or through intrauterine injection, would greatly reduce these risks and alleviate the psychological and financial burdens associated with fertility.

[0003] Ca 2+ As an intracellular second messenger, calcium signaling is widely involved in various physiological processes, including gene expression regulation, synaptic transmission, cell differentiation, cell proliferation, and cell death. Studies have shown that calcium signaling also participates in spermatogenesis, sperm function regulation, and fertilization, making it crucial for male fertility. Sperm are "inert" cells in gene transcription and translation; their physiological functions mainly depend on intracellular second messengers, enzymatic reactions, and post-translational modifications of proteins. Calcium signaling is a regulatory factor in other pathways and therefore plays a central role in sperm function regulation. Existing research evidence, based on the functional analysis of the CatSper calcium channel, demonstrates that the increased calcium signaling caused by CatSper channel-mediated influx of calcium under physiological stimulation is essential for fertilization, and the loss of the major subunit of CatSper can lead to infertility due to sperm dysfunction. However, calcium signaling is not only affected by influx of calcium; the release and reabsorption of calcium from the calcium pool are also important processes regulating calcium signaling, but these have not been fully studied due to the lack of ideal research tools, drugs, or animal models. Furthermore, since mature sperm lose a large number of organelles, the function of the calcium reservoir is mainly undertaken by the acrosome and a small number of mitochondria. Many proteins that regulate the calcium reservoir in somatic cells are absent or lose their function in sperm. However, the IP3R channel is thought to exist in mature sperm and may participate in sperm hyperactivation, but the evidence is insufficient. Therefore, investigating the role of the calcium reservoir in sperm function regulation and fertilization by focusing on this important and complex intracellular calcium channel, IP3R, is of great significance.

[0004] IP3R, or inositol 1,4,5-trisphosphate receptor, is a calcium ion channel located in the endoplasmic reticulum and sarcoplasmic reticulum. Phospholipase C (PLC) catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to form IP3 (inositol 1,4,5-trisphosphate (IP3)) and DAG (diacylglycerol). IP3 is transported intracytoplasm, and its binding to IP3R receptors in the sarcoplasmic or endoplasmic reticulum regulates channel opening and calcium signaling. There are three different types of IP3R channels, which are highly homologous. Different subtypes can also form functional heterotetrameric channels, resulting in greater diversity of IP3R channels, which facilitates their participation in the regulation of a wide range of physiological activities. However, this also leads to compensatory mechanisms between different channels, posing a significant challenge to elucidating the function of individual IP3R channels in specific tissues and organs. Furthermore, inhibitors of the IP3R channel, 2-APB and Xestospongin C, have shown potential therapeutic effects in neurodegenerative diseases (such as Alzheimer's disease) and cardiovascular diseases. Future drug development is focused on finding highly specific IP3R modulators with low side effects, particularly selectively regulating specific subtypes (such as the role of IP3R2 in cardiomyocytes). Whether IP3R can serve as a therapeutic target for male infertility in mature sperm function requires further investigation. All of this research relies heavily on well-developed animal models, as pharmacological inhibition alone may lack specificity, lead to off-target effects, or cause side effects, resulting in insufficient evidence.

[0005] Therefore, there is a lack of animal research models in this field that can analyze the function of mature sperm calcium stores. It is difficult to determine whether the effects caused by calcium store agonists come from specific targets. There is an urgent need for relevant models to prove whether calcium stores have a core function in fertilization, so as to explore whether they can be used as diagnostic or contraceptive targets for male infertility and to serve human reproductive health. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by proposing a method for constructing a gene-edited mouse model for studying the calcium store function in mature sperm and low fertility, and its application. Based on the Cre-LoxP system, a research scheme for analyzing the function of different IP3R subtypes is provided, and pharmacological tools for analyzing IP3R in mature sperm have been identified through pharmacological screening. This invention fills the research gap in the field of reproductive medicine regarding multi-subtype IP3R calcium channels, provides a research scheme and tools for analyzing functional compensation among different IP3R subtypes, and establishes a novel mouse model of low fertility due to calcium signaling disorders. It provides a fundamental development platform and tools for research on diagnostic and therapeutic targets for male infertility based on calcium signaling.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A method for constructing a gene-edited mouse model to study the function of calcium stores in mature sperm and low fertility includes the following steps:

[0009] Step 1: Using IP3R three-site homozygous Loxp mice donated from Professor Ouyang Kunfu's laboratory at Peking University Shenzhen Research Institute, and the B6-Igs2 mice previously invented and constructed by our team... (Ddx4CreERT2) NTUXi mice mating produces three-point heterozygous mice with... Ddx4CreERT2 loxp mice (IP3R1) f / + IP3R2 f / + IP3R3 f / + Ddx4 ERT2 / + );

[0010] Step 2: Further crossbreeding with IP3R 3-site homozygous Loxp mice to obtain target mice for conditional knockout (IP3R1). f / f IP3R2 f / f IP3R3 f / f Ddx4 ERT2 / + It is abbreviated as IP3R-tKO, and the control mouse is named IP3R-Con (IP3R1). f / f IP3R2 f / f IP3R3 f / f );

[0011] Step 3: Finally, adult IP3R-tKO and IP3R-Con were injected intraperitoneally with tamoxifen (2 mg / 30g body weight) for 5 consecutive days to induce Cre enzyme to exert recombination effect. After genotyping and recombination efficiency determination, mice with specific knockout of three subtypes of IP3R in mature sperm were obtained. Finally, after fertility testing, target model mice with sperm dysfunction and low fertility were obtained.

[0012] A gene-edited mouse model obtained using the above-described construction method.

[0013] The present invention also provides an application of the above-described gene-edited mouse model in drug screening for improving fertility.

[0014] Preferably, it is used for mechanism research on fertility-related diseases caused by IP3R calcium signaling disorders, in which a significant increase in spontaneous acrosome response leads to impaired fertilization capacity.

[0015] An application of the gene-edited mouse model described above in the function of calcium stores in mature sperm.

[0016] Preferably, the study of IP3R-related calcium signals in sperm is constructed by screening and testing agonist drugs using a detection platform.

[0017] Preferably, Bradykinin and Thimerosal can significantly increase the calcium signaling response level of the calcium pool in mouse sperm head.

[0018] Preferably, Bradykinin and Thimerosal can be used as evaluation tools for assessing whether the calcium reservoir function in human sperm is abnormal. Specifically, 5 μM Bradykinin and Thimerosal do not affect sperm motility and survival, but significantly increase the calcium signaling response level of the head calcium reservoir.

[0019] Traditionally, Bradykinin and Thimerosal are thought to induce calcium ion release from the calcium pool via the endoplasmic reticulum IP3R calcium channel. However, whether they have this effect in mature sperm is unknown, and whether they are suitable tools for studying sperm calcium signaling remains unclear. Therefore, based on the successful construction of the IP3R-tKO mouse model, this invention further confirms that Bradykinin and Thimerosal can be used to study the function of IP3R in mature sperm. Furthermore, the calcium signaling they induce requires high-resolution single-cell imaging; other calcium signal detection devices such as ELISA readers cannot capture changes in calcium pool signaling.

[0020] By adopting the above technical solution: This invention utilizes the characteristics of tamoxifen-induced gene recombination to achieve simultaneous knockout of three IP3R subtypes in mature sperm. Furthermore, through screening potential IP3R agonist drugs and detection platforms, a research animal model and strategy for studying the calcium store function in mature sperm have been obtained.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. The IP3R-tKO mouse used in this invention can simultaneously knock out the three subtypes of IP3R in mature sperm, which is a first invention and report.

[0023] 2. The IP3R-tKO mice used in this invention exhibited largely normal spermatogenesis, but the main impairment lay in the function of mature sperm, manifested as reduced sperm motility, decreased sperm count, and premature acrosome reaction. This resulted in impaired fertility, characterized by stable low fertility. Further knockout of the remaining IP3R expression in their offspring further reduced fertility, with some mice even becoming completely infertile. These results indicate that this mouse model is a valuable indicator of impaired fertility due to sperm dysfunction and holds promise for future use as a drug evaluation model for improving fertility based on sperm function.

[0024] 3. Based on the conservation of the IP3R channel between humans and mice, this invention is expected to advance research on the diagnosis and treatment of male infertility in humans.

[0025] 4. The IP3R-Con and IP3R-tKO mouse sperm used in this invention showed significant differences in their responses to Bradykinin and Thimerosal stimulation. This provides clear and reliable evidence that the mature sperm calcium storage release channel IP3R plays a role in regulating calcium signaling, making it a good model for studying the function and mechanism of the sperm calcium storage release channel.

[0026] 5. Based on animal model evidence, this invention further utilizes clinical human sperm samples to confirm that Bradykinin and Thimerosal are research tools for studying the role of IP3R-mediated calcium store release. Attached Figure Description

[0027] Figure 1 This document presents the IP3R-tKO mouse construction strategy and successful construction verification results in Example 1 of this invention. A represents the mating and breeding strategy. B represents genotype identification and gene recombination detection. WT (wild type), Fl / + (heterozygote), Flox (homozygote), and △ indicates gene recombination (gene knockout). C represents the expression levels of the three subtypes IP3R1, IP3R2, and IP3R3 in IP3R-tKO mice detected using testes, further confirming successful knockout.

[0028] Figure 2 This is a graph showing the results of evaluating sperm function (motility, acrosome response) and fertilization capacity in IP3R-tKO mice in Example 2 of this invention. A represents the total motility of IP3R-tKO, B represents the forward motility, C represents the sperm concentration, D represents the acrosome response level, and E represents the fertility assessment.

[0029] Figure 3The figures show the results of the study on calcium signaling mediated by the IP3R-tKO calcium pool in this invention. A is an example figure of sperm calcium signaling induced by Thimerosal (Thi) and Bradykinin (BK), B is the time-effect curve corresponding to figure A, and C is a statistical graph of the maximum value of calcium signal response.

[0030] Figure 4 This diagram shows the results of evaluating calcium store response levels in human sperm using Bradykinin and Thimerosal, as described in this invention. A is an example diagram of sperm calcium signaling induced by Bradykinin and Thimerosal; B is the time-effect curve corresponding to diagram A; C is a statistical graph of the maximum calcium signal response. D confirms, using sperm samples from CatSper-deficient patients, that the calcium signal induced by Bradykinin and Thimerosal originates from the calcium store, while the calcium signal induced by ATP as a control originates from CatSper-mediated extracellular calcium influx. Detailed Implementation

[0031] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings, so that those skilled in the art can better understand the advantages and features of the present invention, thereby making a clearer definition of the scope of protection of the present invention. The embodiments described in this invention are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0032] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0033] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0034] Example 1: Construction of IP3R-tKO mouse model with simultaneous and specific knockout of three IP3R subtypes in sperm

[0035] 1. Breeding strategies for IP3R-Con and IP3R-tKO mice are described in [link to documentation]. Figure 1 A. After two rounds of mating, the target mice were obtained through genotyping and screening.

[0036] 2. After obtaining IP3R-Con and IP3R-tKO mice, tamoxifen (2 mg / 30 g body weight) was injected intraperitoneally for 5 consecutive days to induce Cre enzyme to exert its recombinant effect. After 14 days, the recombinant enzyme was fully expressed, and the newly generated sperm after knockout gradually reached maturity, while the original sperm that were not knocked out aged or were excreted through metabolism.

[0037] 3. Gene identification of recombinant mice. Mice tail tissue was excised, DNA was extracted, and PCR products were separated and identified by 2% agarose gel electrophoresis. The primers used for identification are shown in Table 1 below.

[0038] 4. Detection of recombination effect of Ddx4ERT2 Cre recombinase on Flox site. Testicular tissue was obtained from dissected mice, and DNA was extracted. Using primers specifically designed for identifying recombinant DNA products (Table 1), the presence of gene recombination in testicular germ cells was detected. If recombination occurred, a band of XXX bp could be amplified by PCR.

[0039] 5. Simultaneously, RNA was extracted from tamoxifen-induced testicular tissue using a commercially available RNA extraction kit. The expression levels of the three IP3R isoforms mRNA were quantified by qPCR. The primers used for quantification are shown in Table 1. Figure 1 The results showed that the expression levels of three genes were significantly reduced in IP3R-tKO mice, indicating that the gene knockout was successful and the mouse model was successfully constructed.

[0040]

[0041] Example 2: Evaluation of sperm function and fertility in IP3R-tKO mice

[0042] 1. Sperm function evaluation. Validated IP3R-tKO mice were sacrificed along with control mice for sperm function evaluation. Sperm motility was assessed using Hamilton's computer-aided semen analysis system, which measured total motility and progressive motility. Figure 2 A and 2B). Sperm count was determined using a hemocytometer (A and B). Figure 2 C). The acrosome reaction was analyzed using PNA staining. The specific experimental steps are as follows:

[0043] (1) Take sperm from the epididymal tail of mice, add HTF capacitation solution, and capacite at 37°C for 90 min; take the capacited supernatant sperm and add HS, Thi and FF (follicular fluid) respectively, and incubate at 37°C in the dark.

[0044] (2) Centrifuge to remove supernatant and resuspend using HS; add mitochondrial activity indicator dye MitotrackerRed and incubate at 37°C in the dark; after incubation, centrifuge to remove supernatant; resuspend using HS and take an appropriate amount to plate, dry the plate and fix with methanol, then wash with PBST; use acrosome dye PNA, incubate at 37°C for 20 min, wash with PBST and add DAPI to mount the plate.

[0045] (3) Use an upright fluorescence microscope to take pictures, count the proportion of sperm that have undergone acrosome reaction (positive mitochondrial staining + incomplete PNA staining acrosome indicates that sperm have undergone acrosome reaction), and analyze and process the data.

[0046] 2. Fertility Evaluation. To study the fertility of male mice, mice were divided into IP3R-tKO (experimental group) and IP3R-Con (control group), with 5 mice in each group. They were kept together for at least 4 months at a male-to-female ratio of 1:2, and the number of offspring was counted. Because the knockout efficiency of Ddx4ERT2Cre cannot be guaranteed to reach 100%, there may still be IP3R protein that has not been completely knocked out in the sperm. This may have led to the IP3R-tKO mice still being able to reproduce despite a reduced number of offspring in the first round of fertility testing. Therefore, to further improve the gene recombination knockout efficiency, a second round of tamoxifen-induced knockout was performed on the offspring of the already induced IP3R-tKO mice, using the same method. Then, the mice were again divided into IP3R-tKO2 (experimental group) and IP3R-Con2 (control group), with 5 mice in each group. They were kept together for at least 4 months at a male-to-female ratio of 1:2, and the number of offspring was counted. At this point, it was found that the fertility of the mice was further reduced, and some mice were even completely infertile. Figure 2 E).

[0047] Example 3: Study on IP3R-mediated calcium signaling in the calcium pool of IP3R-tKO mice

[0048] 1. Separate mouse epididymal tail sperm into 1 mL HS solution for 10 min;

[0049] 2. Use micro forceps to remove the tissue block, aspirate 5 μL of sperm and observe it under a microscope to ensure a survival rate of over 85%;

[0050] 3. Stain with 2 μM Calbryte 520 AM (AAT Bioquest, Inc., Sunnyvale, CA, USA) and 0.02% F-127 (Beyotime, Shanghai, China) at 37℃ in the dark for 30 min.

[0051] 4. Add 100 μL of sperm to a sperm plating dish and fix the sperm for 10 min;

[0052] 5. After washing with calcium-containing HBSS 2-3 times, use a calcium imaging instrument for testing. After confirming sperm viability (continuous tail wagging), add drug stimulation and take pictures, with a 2-second interval. First, take 30-60 seconds of basic fluorescence before adding drug stimulation, and take a total of 200 pictures.

[0053] 6. ImageJ processes data.

[0054] Example 4:

[0055] Bradykinin and Thimerosal were used to evaluate the calcium signaling response levels of the calcium pool in clinical human sperm samples to screen specific infertile populations whose sperm dysfunction is caused by abnormal calcium pool function, and to further investigate the mechanism. In a calcium-rich environment, intracellular calcium pool release was induced in sperm from CatSper-deficient patients and normal volunteers (Normal Sperm, NS) using Thimerosal (Thi) and Adenosine Triphosphate (ATP), respectively. In a calcium-free environment, intracellular calcium pool release was induced in normal volunteers (Normal Sperm, NS) using Thimerosal (Thi), Bradykinin (BK), and Adenosine Triphosphate (ATP). It was found that the agonists Thi and BK significantly induced calcium signaling (CatSper2 mutation and external Ca2+) that increased calcium ion release from the calcium pool. 2+ Free condition) ( Figure 4 ATP, like the control group HS, did not induce an increase in calcium signaling. The specific experimental steps are as follows:

[0056] 1. In advance, embed the culture dish with 80 μL of 0.05% poly-L-lysine, canavalia protein, or 3% Cell-tak, and dry it at 55℃ before use.

[0057] 2. Take a suitable sample and wash it twice with calcium-containing HS.

[0058] 3. Resuspend sperm in HS, adjust the final sperm concentration to approximately 10^6 / mL, and stain with 2 μM Fluo-4 AM, 0.05% F-127, 37℃, in the dark for 30 min.

[0059] 4. Centrifuge at 300g for 6 minutes, wash twice with calcium-containing HS, and wash the coated petri dishes twice at the same time.

[0060] 5. Add 100 μL of sperm plating dish and fix the sperm for 10 min;

[0061] 6. After washing twice with calcium-free HS, use calcium imaging for detection. Take pictures at 2-second intervals. First, take 30-60 seconds of basal fluorescence, then add Thi, BK or ATP stimulation. Take a total of 250-300 pictures.

[0062] 7. ImageJ processes data.

[0063] In summary, this invention utilizes tamoxifen-induced, Ddx4ERT2 promoter-driven Cre recombinase expression to specifically and simultaneously knock out three subtypes of IP3R in sperm, eliminating compensatory effects between subtypes, and is used to study its role and mechanism in male fertility. After two rounds of induction and knockout, the mouse model exhibited severely impaired fertility, reduced sperm count and motility, and abnormal acrosome reaction, making it a good model for evaluating sperm dysfunction. It is hoped that this model can be used in the future to screen for therapeutic drugs to improve sperm dysfunction, especially male infertility caused by premature acrosome reaction. Furthermore, through screening and optimization of IP3R agonists and calcium signal detection platforms, this mouse model was further confirmed as a good model for studying calcium signaling mediated by sperm calcium stores. The reliability of Bradykinin and Thimerosal in studying the release of IP3R-mediated calcium from sperm was further confirmed in clinical human sperm. This provides research methods, technical platforms, and animal models for elucidating the role and regulatory mechanisms of calcium stores in mature sperm during fertilization, effectively filling the gap in the field of sperm calcium store research due to the lack of experimental animal models and reliable tools and drugs.

[0064] The descriptions and practices disclosed in this invention are readily apparent and understandable to those skilled in the art, and various modifications and refinements can be made without departing from the principles of this invention. Therefore, any modifications or improvements made without departing from the spirit of this invention should also be considered within the scope of protection of this invention.

Claims

1. A method for constructing a gene-edited mouse model to study the function of calcium stores in mature sperm and low fertility, characterized in that, Includes the following steps: Step 1: Using IP3R three-site homozygous Loxp mice and constructed B6-Igs2 (Ddx4CreERT2) NTUXi mice mating produces three-point heterozygous mice with... Ddx4CreERT2 loxp mice; Step 2: Further crossbreed with IP3R three-point homozygous Loxp mice to obtain target mice for conditional knockout, which are abbreviated as IP3R-tKO, and the control mice are named IP3R-Con; Step 3: Finally, adult IP3R-tKO and IP3R-Con were injected intraperitoneally with tamoxifen for 5 consecutive days to induce Cre enzyme to exert recombination effect. After genotyping and recombination efficiency determination, mice with specific knockout of three subtypes of IP3R in mature sperm were obtained. Finally, after fertility testing, target model mice with sperm dysfunction and low fertility were obtained.

2. The gene-edited mouse model obtained by the construction method according to claim 1.

3. The application of the gene-edited mouse model according to claim 2 in drug screening for improving fertility.

4. The application according to claim 3, characterized in that, This study investigates the mechanism by which IP3R calcium signaling disorders lead to fertility-related diseases.

5. The application of the gene-edited mouse model according to claim 2 in the calcium store function of mature sperm.

6. The application according to claim 5, characterized in that, A study on IP3R calcium signaling in sperm was conducted using a screening and detection platform for agonist drugs.

7. The application according to claim 5, characterized in that, Bradykinin and Thimerosal significantly increased the calcium signaling response level of the calcium pool in mouse sperm heads.

8. The application according to claim 6, characterized in that, Bradykinin and Thimerosal are used as tools to evaluate whether the calcium store function in human sperm is abnormal.

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