Application of IPO5 as male infertility diagnostic marker

Abstract

The invention relates to the technical field of biological medicine, in particular to application of IPO5 as a male infertility diagnostic marker. The distribution and expression of IPO5 in sperms are detected by using immunofluorescence staining and western blot, IPO5 in normal test body sperms is distributed at the neck and head of the sperms, a small amount of IPO5 in oligospermia and asthenospermia test body sperms is distributed at the head of the sperms, IPO5 expression in sperms of oligospermia and asthenospermia test bodies is abnormal, and IPO5 can be used as a marker for detecting male infertility. And a foundation is laid for clinical diagnosis of male infertility diseases such as oligospermia and asthenospermia.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to the use of IPO5 as a diagnostic marker for male infertility. Background Art

[0002] About 15% of couples worldwide face infertility, of which male factors contribute as much as 40%-50%. Unhealthy lifestyle habits (such as sitting for long periods of time and smoking), environmental pollution, and social pressure have led to a continuous decline in male sperm quality. Chinese male semen parameters have declined significantly in the past 40 years, with sperm concentration decreasing by an average of 32% and motility decreasing by 27%. Male infertility is no longer a simple reproductive disorder, but a complex health problem involving multiple factors such as genetics, endocrine system, and psychology. Although assisted reproductive technology (ART) provides patients with oligoasthenospermia with the possibility of fertility, its success rate is still low and there are many uncertainties. More and more evidence shows that offspring conceived through ART technology may face a higher risk of multiple diseases. Therefore, improving male sperm quality is urgent.

[0003] Clinical evaluation of semen quality mainly relies on indicators such as sperm motility and quantity, which can preliminarily judge the possibility of natural conception and assist in deciding whether ART is needed. However, external factors such as environmental factors, sleep quality, and work and rest habits affect semen parameters, leading to misjudgment and even delaying the timing of treatment. For the decline in semen quality caused by genetic factors such as spermatogenesis disorders, more accurate diagnostic methods are needed. In addition, the current detection methods for sperm dysfunction caused by single gene mutations or abnormal expression are still imperfect. IPO5 stands for Importin 5, also known as karyopherin beta-3 (KPNB3). The present invention compares the expression and distribution differences of IPO5 in the semen of normal men and patients with oligoasthenospermia, and determines that abnormal IPO5 expression can be used as a potential biomarker for diagnosing oligoasthenospermia, providing a new strategy for clinical male infertility diagnosis. Summary of the Invention

[0004] The purpose of the present invention is to solve the deficiencies in the prior art and to propose an application of IPO5 as a diagnostic marker for male infertility.

[0005] In order to achieve the above object, the present invention is achieved through the following technical solutions:

[0006] The invention relates to the use of IPO5 as a marker in the preparation of a kit for diagnosing male infertility, wherein the kit detects whether there is a disorder in the sperm function of the subject by testing the level of IPO5 in the semen sample of the subject.

[0007] The subject is a mammal or a human. Targeting the protein sequence, antibodies against the IPO5 antigen are immunized against amino acids 1-300 of karyopherin beta-3 from humans, mice, and rats.

[0008] Furthermore, the level of IPO5 in the semen sample of the subject is tested by: detecting the distribution of IPO5 in the sperm of the subject by immunofluorescence staining;

[0009] Alternatively, Western blotting was used to detect the expression of IPO5 in spermatozoa of the subjects.

[0010] Furthermore, the kit includes paraformaldehyde, TritonX-100, Bovine Serum Albumin, mouse anti-IPO5 antibody, anti-mouse 568 antibody, diluent TBST, and DAPI dye.

[0011] Furthermore, the kit includes RIPA, protease inhibitors, protein markers, gel, methanol, PVDF membrane, mouse anti-IPO5, mouse anti-Gapdh, anti-mouse 680, and diluent TBST.

[0012] The kit includes paraformaldehyde, TritonX-100, Bovine Serum Albumin, mouse anti-IPO5 antibody, anti-mouse 568 antibody, diluent TBST, and DAPI dye.

[0013] Furthermore, the criteria for determining whether there is a disorder in the sperm function of the subject are as follows: if IPO5 in the sperm of the subject is distributed in the sperm neck and head, the subject is judged to be normal; if a small amount of IPO5 in the sperm of the subject is distributed in the sperm head, the subject is judged to be an oligospermic subject.

[0014] If the expression of IPO5 in the semen of the subject is abnormal, the subject is judged as oligoasthenospermic subject, otherwise it is a normal subject.

[0015] Abnormal IPO5 expression includes mutation, deletion or reduction.

[0016] Preferably, the immunofluorescence staining test is performed as follows: semen from normal men (13 cases) and men with oligoasthenospermia (11 cases) is collected, and the collected semen is respectively concentrated into 15 ml centrifuge tubes, a small amount is taken for mirror smear, and the rest is centrifuged and frozen;

[0017] Use a pipette to evenly apply 10 μl of semen to a slide coated with 0.1% gelatin. Once dry, proceed to immunofluorescence staining. Immunofluorescence staining involves fixation, permeabilization, blocking, primary antibody incubation, secondary antibody incubation, and nuclear staining.

[0018] Fixation: The obtained sperm smear was exposed to 4% paraformaldehyde and incubated at room temperature for 15 minutes;

[0019] Permeabilization: Expose sperm to 0.1% TritonX-100 and incubate at room temperature for 10 minutes;

[0020] Blocking: Expose sperm to 5% Bovine Serum Albumin (BSA) and incubate at room temperature for 60 minutes;

[0021] Primary antibody incubation: Spermatozoa were exposed to mouse anti-IPO5 antibody diluent (TBST) at a dilution ratio of 1:200 at 4°C overnight.

[0022] Secondary antibody incubation: Spermatozoa were exposed to anti-mouse 568 antibody diluent (TBST) at a dilution ratio of 1:500 and incubated at room temperature for 60 minutes.

[0023] Nuclear staining: Spermatozoa were exposed to 5 μg / ml DAPI dye and incubated at room temperature for 5 minutes.

[0024] Furthermore, the level of IPO5 in the semen sample of the subject is tested by detecting the expression of IPO5 in the sperm of the subject using protein immunoblotting.

[0025] The kit includes RIPA, protease inhibitors, protein markers, gel, methanol, PVDF membrane, mouse anti-IPO5, mouse anti-Gapdh, anti-mouse 680, and diluent TBST.

[0026] Preferably, the specific steps of detecting the expression of IPO5 in the sperm of the subject by Western blotting are as follows:

[0027] Sperm was collected and transferred to a 1.5 ml centrifuge tube. The semen was centrifuged at 1500 rpm for 5 minutes, snap-frozen in liquid nitrogen, and stored at -80°C. Western blotting analysis was performed, including protein lysis, protein electrophoresis, membrane transfer, blocking, primary antibody incubation, and secondary antibody incubation.

[0028] Protein lysis: The sperm pellet was lysed using RIPA, with protease inhibitors pre-added to RIPA. After placing on ice for 30 minutes, centrifuge at 14,000 rpm for 10 minutes and transfer the supernatant to a new 1.5 ml centrifuge tube. After measuring the protein concentration, add Omni-Easy TM Instant protein loading buffer, boil at 95℃ for 5 minutes and place on ice until use.

[0029] Protein electrophoresis: Remove the denatured protein sample after lysis and centrifuge briefly. Add 5 μl of protein marker and 15 μl of protein sample from different groups to the gel. Electrophoresis at 120V for 90 minutes.

[0030] Transfer: First, activate the PVDF membrane with methanol. After 10 seconds, rinse with water. Assemble the transfer apparatus in a sandwich configuration. Add pre-chilled transfer buffer and place the transfer chamber in an ice box. Connect the power supply and set a constant current of 300mA for 60 minutes.

[0031] Blocking: Remove the PVDF membrane and wash it once with TBST buffer. Place it in an incubation box pre-filled with 5% BSA buffer and shake it slowly on a shaker at room temperature for 1 hour.

[0032] Primary antibody incubation: Add primary antibodies (mouse anti-IPO5 diluted in TBST at a ratio of 1:500; mouse anti-Gapdh diluted in TBST at a ratio of 1:2000), incubate at 4°C overnight, and wash the membrane three times with TBST for 5 minutes each time.

[0033] The epitope of the primary antibody IPO5 is located at amino acids 1-300 of the N-terminus;

[0034] Secondary antibody incubation: Add secondary antibody (anti-mouse 680 diluted 1:20,000 in TBST) and incubate at room temperature in the dark for 1 hour. Wash the membrane three times with TBST for 5 minutes each.

[0035] The present invention has the following beneficial effects: the present invention collects semen from normal men and men with oligoasthenospermia, and uses immunofluorescence staining and protein immunoblotting to examine the distribution and expression of IPO5 in sperm. In the sperm of normal subjects, IPO5 is distributed in the sperm neck and head, while in the sperm of oligoasthenospermia subjects, IPO5 is distributed in a small amount in the sperm head. It is clarified that the expression of IPO5 is decreased in the semen of oligoasthenospermia, and IPO5 is proposed as a marker for detecting male infertility, laying a foundation for the clinical diagnosis of male infertility diseases such as oligoasthenospermia. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Immunofluorescence staining indicates the distribution of IPO5 in semen of normal men and men with oligoasthenospermia;

[0037] a. Distribution of IPO5 in sperm of normal men; b. Distribution of IPO5 in sperm of men with oligoasthenozoospermia. Red indicates IPO5, and blue indicates the cell nucleus.

[0038] Figure 2 Western blotting was used to compare the expression of IPO5 in spermatozoa of normal men and men with oligoasthenospermia;

[0039] a. Western blotting analysis of IPO5 and Gapdh in spermatozoa of normal and oligoasthenozoospermic men; b. Statistical analysis of grayscale values ​​of Western blotting bands. DETAILED DESCRIPTION

[0040] The present invention will be further described below with reference to the examples, but they are not intended to limit the present invention.

[0041] The raw materials used are as follows:

[0042] Paraformaldehyde (Biyuntian Biotechnology)

[0043] TritonX-100 (Biyuntian Biotechnology)

[0044] Bovine Serum Albumin (BSA, Sigma)

[0045] DAPI dye (Sigma)

[0046] RIPA (Biyuntian Biotechnology)

[0047] Protease inhibitors (Apexbio)

[0048] Omni-Easy TM Instant protein loading buffer (Yazyme Bio)

[0049] mouse anti-IPO5 (Santa Cruz)

[0050] mouse anti-Gapdh (Sigma)

[0051] anti-mouse 680 (Invitrogen)

[0052] 1. Comparison of the distribution of IPO5 in sperm of normal men and oligoasthenospermia men by immunofluorescence staining

[0053] Semen was collected from 13 healthy men and 11 men with oligoasthenospermia and transferred to 15ml centrifuge tubes. Using a pipette, 10μl of semen was evenly smeared onto a pre-coated glass slide. Immunofluorescence staining was then performed. Finally, the sperm smear was observed under a fluorescence microscope and images were obtained.

[0054] Specifically:

[0055] Use a pipette to draw 10 μl of semen and evenly smear it on a slide coated with 0.1% gelatin in advance. After drying, proceed to immunofluorescence staining steps:

[0056] (1) Fixation: The obtained sperm smear was exposed to 4% paraformaldehyde and incubated at room temperature for 15 minutes;

[0057] (2) Permeabilization: Expose sperm to 0.1% TritonX-100 and incubate at room temperature for 10 minutes;

[0058] (3) Blocking: Expose sperm to 5% Bovine Serum Albumin (BSA) and incubate at room temperature for 60 minutes;

[0059] (4) Primary antibody incubation: Spermatozoa were exposed to mouse anti-IPO5 antibody at a dilution ratio of 1:200 at 4°C overnight;

[0060] (5) Secondary antibody incubation: Spermatozoa were exposed to anti-mouse 568 antibody at a dilution ratio of 1:500 and incubated at room temperature for 60 minutes;

[0061] (6) Nuclear staining: Spermatozoa were exposed to 5 μg / ml DAPI dye and incubated at room temperature for 5 minutes.

[0062] 2. Western blotting to compare the expression of IPO5 in spermatozoa of normal men and oligoasthenozoospermia men

[0063] After centrifugation, the semen of normal men and men with oligoasthenospermia is frozen or subjected to protein lysis. First, add 50-100 μl of RIPA lysis buffer, to which an appropriate amount of protease inhibitors are pre-added, including 1% PMSF, 0.1% DTT and 0.1% phosphorylation protease inhibitors; place on an automatic grinder for protein lysis. Subsequently, centrifuge briefly and place on ice for 30 minutes. After centrifugation at 14000 rpm for 10 minutes, transfer the supernatant to a new 1.5 ml centrifuge tube. After measuring the protein concentration, add Omni-Easy TM Prepare instant protein loading buffer, boil at 95°C for 5 minutes, and place on ice until ready to use. Prepare a 10% separation gel for electrophoresis, transfer to the membrane, block the membrane, and incubate with primary and secondary antibodies. Place the PVDF membrane in an imaging system and acquire images.

[0064] Test results:

[0065] 1. Distribution of IPO5 in semen of normal men and men with oligoasthenospermia

[0066] like Figure 1 As shown in Figure 2, the distribution of IPO5 in sperm was examined. The results showed that in normal male sperm, IPO5 was mainly expressed in the sperm neck and head, while in oligoasthenozoospermia patients, a small amount of IPO5 was distributed in the sperm head, suggesting that compared with normal men, the distribution of IPO5 in the sperm of oligoasthenozoospermia men is abnormal.

[0067] 2. Expression of IPO5 in semen of normal men and men with oligoasthenospermia

[0068] like Figure 2 As shown in Figure 2, Western blotting was used to compare the expression of IPO5 in spermatozoa of normal men and men with oligoasthenozoospermia. The results showed that the expression of IPO5 in spermatozoa of men with oligoasthenozoospermia was significantly decreased compared with that of normal men (0.55±0.99 vs 0.24±0.05, P<0.01).

[0069] The above shows and describes the basic principles, main features, and advantages of the present invention. However, the above is only a specific embodiment of the present invention, and the technical features of the present invention are not limited thereto. Any other implementation methods derived by any person skilled in the art without departing from the technical solution of the present invention should be included in the patent scope of the present invention.

Claims

1. Use of IPO5 as a marker in the preparation of a kit for diagnosing male infertility, characterized in that: The kit detects whether there is a disorder in the sperm function of the subject by testing the level of IPO5 in the semen sample of the subject.

2. The use according to claim 1, characterized in that The subject is a mammal.

3. The use according to claim 1, characterized in that The subject is a human.

4. The use according to claim 1, characterized in that The level of IPO5 in the semen sample of the test subject is specifically tested by detecting the distribution of IPO5 in the sperm of the test subject by immunofluorescence staining.

5. The use according to claim 1, characterized in that The level of IPO5 in the semen sample of the subject was tested by specifically detecting the expression of IPO5 in the sperm of the subject using protein immunoblotting.

6. The use according to claim 1, characterized in that The kit includes paraformaldehyde, TritonX-100, Bovine Serum Albumin, mouse anti-IPO5 antibody, anti-mouse 568 antibody, diluent TBST, and DAPI dye.

7. The use according to claim 1, characterized in that The kit includes RIPA, protease inhibitors, protein markers, gel, methanol, PVDF membrane, mouse anti-IPO5, mouse anti-Gapdh, anti-mouse 680, and diluent TBST.

8. The use according to claim 1, characterized in that The criteria for determining whether the sperm function of the subject is impaired are as follows: if IPO5 in the sperm of the subject is distributed in the sperm neck and head, the subject is judged to be normal; if a small amount of IPO5 in the sperm of the subject is distributed in the sperm head, the subject is judged to be an oligospermic subject.

9. The use according to claim 1, characterized in that The criterion for determining whether the sperm function of the subject is impaired is: if the expression of IPO5 in the semen of the subject is abnormal, the subject is judged to be an oligoasthenospermic subject; otherwise, the subject is normal.

10. The use according to claim 9, characterized in that Abnormal IPO5 expression includes mutation, deletion or reduction.

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