NL2038543A - Rat testicular progenitor leydig cell marker protein, screening method and use thereof - Google Patents
Rat testicular progenitor leydig cell marker protein, screening method and use thereof Download PDFInfo
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Abstract
Provided are a rat testicular progenitor Leydig cell marker protein, a screening method and use thereof. In the present disclosure, male rats are treated with EDS to prepare a testicular Leydig cell development model;testicles are collected from the development model 1 week and 3 weeks after treatment with EDS, and interstitium was isolated from testis and digested into single cells for single-cell transcriptomic sequencing to screen differentially expressed genes in Leydig progenitor cells; testicles are isolated from SD male rats of 1-8 weeks old, rat embryos from SD pregnant rats at 15-21 days of gestation and rats on the first day of birth as objects; the RT—qPCR, Western blotting and immunohistochemical testing are performed_for verification and screening to obtain the rat testicular progenitor Leydig cell marker proteins, including SLC38A4, CPA1 and SRD5A1, which can be used to identify or screen rat testicular progenitor Leydig cells.
Description
RAT TESTICULAR PROGENITOR LEYDIG CELL MARKER PROTEIN,
SCREENING METHOD AND USE THEREOF
[0001] The present disclosure belongs to the field of biomedical technology, and particularly relates to a rat testicular progenitor Leydig cell marker protein, a screening method and use thereof.
[0002] Testicular Leydig cells, also known as testicular interstitial cells, mainly function to synthesize androgens and insulin-like factor 3 (INSL3). The adult Leydig cells (ALCs) play a crucial role in the development of the male reproductive system, maintenance of adult reproductive functions and spermatogenesis. ALCs mainly develop from stem
Leydig cells (stem LC, SLC} during adolescence. The process and regulatory mechanism of SLC to ALC maturation are still not well known. In Sprague Dawley (SD) rats, SLCs have differentiated into testicular progenitor Leydig cells (PLCs) at 3 weeks of age, developed into immature Leydig cells (ILCs) at 5 weeks of age, and finally became Thoroughly mature ALCs at 8 weeks of age (see Figure 1). PLC is a critical intermediate step in the development of testicular Leydig cells. Therefore, the development of PLCs reflects the number and development of testicular Leydig cells to some extent.
[0003] The research on the biological functions of PLCs is very important for the study of its differentiation, identification and proliferation. The development of cell-specific marker proteins is an effective approach to understand the number and proliferation of corresponding cells. Unfortunately, however, the cellular characteristics, marker proteins and regulatory mechanisms of PLCs at different stages are still unclear.
[0004] In view of this, an object of the present disclosure is to provide a rat testicular progenitor Leydig cell marker protein, and screen three proteins that specifically appear in the PLC development stage during ALC development through single-cell sequencing, qPCR, western blotting and immunohistochemistry, which can be used as PLC marker proteins.
[0005] The present disclosure provides a rat testicular progenitor Leydig cell marker protein comprising more than one or two of the following proteins: SLC38A4, CPAl and SRD5AL1.
[0006] Preferably, an amino acid sequence of the SLC38A4 is shown in SEQ ID NO: 1.
[0007]Preferably, an amino acid sequence of the CPAl is shown in SEQ ID NO: 2.
[0008] Preferably, an amino acid sequence of the SRDSA1 is shown in SEQ ID NO: 3.
[00092] The present disclosure provides a method for screening the rat testicular progenitor Leydig cell marker protein, comprising the following steps:
[0010] treating male Sprague Dawley (SD) rats with ethylene dimesulfonate (EDS) to prepare a testicular Leydig cell development model, collecting testicles from the development model 1 week and 3 weeks after treatment with EDS, collecting testicles from the development model 1 week and 3 weeks after treatment with ethylene dimesulfonate, isolating interstitium from testis and digestion into single cells for single-cell transcriptomic sequencing to screen differentially expressed genes in Leydig progenitor cells; [0Cl1]isolating testicles using SD male rats of 1 to 8 weeks old, rat embryos from SD pregnant rats at 15 to 21 days of gestation and rats on the first day of birth as objects, performing RT-gPCR to verify the differentially expressed genes using testicular tissues as test materials, and carrying out Western blotting and immunohistochemical testing to verify proteins corresponding to the differentially expressed genes; when the verification result are consistent, using the protein corresponding to the differentially expressed genes in the testicular progenitor
Leydig cells as the rat testicular progenitor Leydig cell marker protein.
[0012] Preferably, the differentially expressed genes in the testicular progenitor Leydig cells comprise Slc38a4, Cpal and
Srdbal;
[0013]Preferably, a PCR primer of the Slc38a4 comprises a forward primer with a nucleotide sequence shown in SEQ ID NO:4 and a reverse primer with a nucleotide sequence shown in
SEQ ID NO:5;
[0014]A gPCR primer of the Cpal comprises a forward primer with a nucleotide sequence shown in SEQ ID NO:6 and a reverse primer with a nucleotide sequence shown in SEQ ID NO:7;
[0015]A gPCR primer of the Srdbal comprises a forward primer with a nucleotide sequence shown in SEQ ID NO:8 and a reverse primer with a nucleotide sequence shown in SEQ ID NO:9.
[0016] The present disclosure provides use of the rat testicular progenitor Leydig cell marker protein or the rat testicular progenitor Leydig cell marker protein obtained by the screening method in identifying or screening rat testicular progenitor Leydig cells.
[0017]The present disclosure provides a rat testicular progenitor Leydig cell marker protein, which comprises at least two of the following proteins: SLC38A4, CPAl and SRDSA1.
According to the present disclosure, rats are treated with
EDS as an ALC development model. Testes are removed 1 week and 3 weeks after treatment with EDS, and cells in the isolated testes are collected for single-cell sequencing to screen for specifically expressed genes at the PLC stage; meanwhile, testes are isolated from SD male rats of 1 to 8 weeks old, rat embryos from SD pregnant rats at 15 to 21 days of gestation and rats on the first day of birth as objects, and the specific genes screened from EDS single-cell sequencing are verified by RT-PCR, WB and immunohistochemistry. The results show that, the single-cell sequencing analysis reveals that ALCs disappear 1 week after treatment with EDS, and Leydig cells
(PLC stage) appear 3 weeks later. In addition to expression of steroid synthesis genes, specific expression genes such as Slc38a4, Cpal and Srdbal are also transiently highly expressed in the PLC stage; besides, the results of immunohistochemistry and western blotting are consistent with the results of RT-qPCR. Therefore, SLC38A4, CPAl and
SRDSA1 have good specificity and sensitivity and can be used as rat testicular progenitor Leydig cell marker proteins for subsequent identification and testing.
[0018]FIG. 1 is a flow chart of the development process of adult Leydig cells (ALCs) in the background art of the present disclosure;
[0012] FIG. 2 is a flow chart of a method for screening the rat testicular progenitor Leydig cell marker protein of the present disclosure;
[0020]FIG. 3 shows the single-cell sequencing results;
[0021]FIG. 4 shows the results of PLC cells expressing various marker genes;
[0022]FIG. 5 shows verification results of each marker gene by RT-gPCR; wherein A is the verification result of gene
Cypl7al, B is the verification result of gene Cypllal; Cis the verification result of gene Srdbal; D is the verification result of gene Cpal, E is the verification result of gene D1k1,
F is the verification result of gene Slc38ad4;
[0023]FIG. 6 shows the verification results by Western blotting;
[0024]FIG. 7 is a statistical diagram of the verification results by Western blotting; wherein, the left figure shows the statistical results of CPAl protein expression, the middle figure shows the statistical results of SLC38A4 protein expression, and the right figure shows the statistical results of CYP17A1 protein expression;
[0025]FIG. 8 shows the immunohistochemistry results of CPAl1.
[0026] The present disclosure provides a rat testicular progenitor Leydig cell marker protein, which comprises more than one or two of the following proteins: SLC38A4, CPAl and 5 SRDSA1.
[0027]In the present disclosure, the amino acid sequence of
SLC38A4 is preferably shown in SEQ ID NO:1 (MDPIELRSVNIEPYEDSCSVDSIQSCYTGMGNSEKGAMDSQFANEDAES QKFLTN
GFLGKKTLTDYADEHHPGTTSFGMSSENLSNAIMGSGILGLSYAMANTGIVLEFVIML
LTVAILSLYSVHLLLKTAKEGGSLIYEKLGEKAFGWPGKIGAFISITMONIGAMSSY
LPIIKYELPEVIRVEMGLEENTGEWYLNGNYLVLEVSVGIILPLSLLKNLGYLGYTS
GESLTCMVFFVSVVIYKKFQIPCPLPVLDHNNGNLTENNTLPMHVIMLPNNSESTGM
NFMVDYTHRDPEGLDEKPAAGPLHGSGVEYEAHSGDKCQPKYFVENSRTAYATPILA
FAFVCHPEVLPIYSELKDRSRRKMOTVSNISITGMLVMYLLAALFGYLSFYGEVEDE
LLHAYSKVYTEDTALLMVRLAVLVAVTLTVPIVLEPIRTSVITLLEPRRPESWVKHF
GIAAIIIALNNVLVILVPTIKYIFGFIGASSATMLIFILPAAFYLKLVKKEPLRSPQ
KIGALVFLVTGIIFMMGSMALITIDWIYNPPNPDHH) . The amino acid sequence of CPAl is preferably shown in SEQ ID NO:2 (MKRLLILSLLLEAVCGNENF VGHQVLRISAADEAQVQKVKELEDLEHLQLDEWRD
AARAGIPIDVRVPFPSIQSVKAFLEYHGISYEIMIEDVQOLLLDEEKOOMSAFQARAL
STDSENYATYHTLDEIYEFMDLLVAEHPQOLVSKIQIGNTFEGRPIHVLKEFSTGGTNR
PAIWIDTGIHSREWVTQASGVWFAKKITKDYGODPTEFTAVLDNMDIFLEIVTNPDGFE
AYTHKTNRMWRKTRSHTOGSLCVGVDPNRNWDAGEGMAGASSNPCSETYRGKEPNSE
VEVKSIVDEVTSHGNIKAFISIHSYSQLLLYPYGYTSEPAPDQAELDQLAKSAVTAL
TSLHGTKEFKYGSIIDTIYQASGSTIDWTYSQGIKYSFTFELRDTGLRGFLLPASQII
PTAEETWLALLTIMDHTVKHPY) . The amino acid sequence of SRD5A is preferably shown in SEQ ID NO:3 (MELDELCLLD MLVYLEGEFMAFVSIVGLRSVGSPYGRYSPQWPGIRVPARPAWFIGQ
ELPSMAWPLYEYIRPAAARLGNLPNRVLLAMELIHYVORTLVEPVLIRGGKPTLLVT
FVLAFLEFCTENGYVOSRYLSQFAVYAEDWVTHPCFLTGEALWLVGMVINIHSDHILR
NLRKPGETGYKIPRGGLFEYVSAANYFGELV EWCGFALASWSLQGVVFALPF TLST
LLTRAKQHHQWYHEKFEDYPKSRKIL IPFVL).
[0028]The present disclosure provides a method for screening the rat testicular progenitor Leydig cell marker protein, comprising the following steps:
[0029]treating male rats with ethylene dimesulfonate (EDS) to prepare a testicular Leydig cell development model,
collecting testicles from the development model 1 week and 3 weeks after treatment with EDS, isolating interstitium from testis and digestion into single cells for single-cell transcriptomic sequencing to screen differentially expressed genes in Leydig progenitor cells;
[0030]iscolating testicles using SD male rats of 1 to 8 weeks old, rat embryos from SD pregnant rats at 15 to 21 days of gestation and rats on the first day of birth as objects, performing RT-gPCR to verify the differentially expressed genes using testicular tissues as test materials, and carrying out Western blotting and immunohistochemical testing to verify proteins corresponding to the differentially expressed genes; when the verification result are consistent, using the protein corresponding to the differentially expressed genes in the testicular progenitor
Leydig cells as the rat testicular progenitor Leydig cell marker protein.
[0031]In the present disclosure, male rats are treated with
EDS to prepare a testicular Leydig cell development model; testicles are collected from the development model 1 week and 3 weeks after treatment with EDS and interstitium was isolated from testis and digested into single cells for single-cell transcriptomic sequencing to screen differentially expressed genes in Leydig progenitor cells.
[0032]In the present disclosure, EDS is used to treat male rats to cause apoptosis of testicular Leydig cells of adult male rats, and promote the development of stem Leydig cells into Leydig cells in the testicles of adult male rats, forming the ALC developmental model. The treatment method of EDS is preferably injection. The concentration of treatment with EDS is preferably 75-85 mg/kg.
[0033]In the present disclosure, 1 week after the male rats are treated with the EDS, stem Leydig cells have begun to develop in the rat testicles. According to the present disclosure, cells isolated from Leydig in the testicle are used as materials for single-cell sequencing. The method of single-cell sequencing is not particularly limited, and any
: single-cell sequencing method well known in the art can be used. In the embodiments of the present disclosure, the single-cell sequencing is completed by Beijing Novogene Co.,
Ltd. In the present disclosure, testes of male rats 1 week and 3 weeks after treatment with EDS are used as materials to perform differential gene analysis on the results of single-cell sequencing, and screen differentially expressed genes in progenitor Leydig cells. The differential gene analysis is completed by Novogene. The differentially expressed genes in the testicular progenitor Leydig cells preferably comprise Slc38a4, Cpal and Srdbal.
[0034]Testes are isolated from SD male rats of 1 to 8 weeks old, rat embryos from SD pregnant rats at 15 to 21 days of gestation and rats on the first day of birth as objects. The
RT-gPCR is performed to verify the differentially expressed genes using testicular tissues as test materials, and the
Western blotting and immunohistochemical testing are carried out to verify proteins corresponding to the differentially expressed genes; when the verification result are consistent, the protein corresponding to the differentially expressed genes in the testicular progenitor Leydig cells is used as the rat testicular progenitor Leydig cell marker protein.
[0035]In the present disclosure, SD male rats of 1 to 8 weeks old include SD male rats of 1, 2, 3, 4, 5, 6, 7, and 8 weeks old, which correspond to stem Leydig cells, progenitor Leydig cells, immature Leydig cells and various development stages of Leydig cells respectively. Rat embryos from SD pregnant rats at 15 to 21 days of gestation include embryos at 15, 16, 17, 18, 19, 20 and 21 days of gestation and SD rats on the first day of birth, which correspond to stem Leydig cells, progenitor Leydig cells, immature Leydig cells and various development stages of Leydig cells in the embryonic stage, respectively.
[0036]In the present disclosure, the gPCR primer of the
Slc38a4 preferably comprises a forward primer with a nucleotide sequence shown in SEQ ID NO:4 and a reverse primer with a nucleotide sequence shown in SEQ ID NO:5. The qPCR primer of the Cpal preferably comprises a forward primer with a nucleotide sequence shown in SEQ IDNO: 6 and a reverse primer with a nucleotide sequence shown in SEQ ID NO:7. The gPCR primer of the Srdbal preferably comprises a forward primer with a nucleotide sequence shown in SEQ ID NO:8 and a reverse primer with a nucleotide sequence shown in SEQ ID NO:9. In the present disclosure, the methods of Western blotting and immunohistochemical testing are not particularly limited, and any Western blotting and immunohistochemical testing methods well known in the art can be used.
[0037]In the embodiments of the present disclosure, the gPCR test results show that Slc38a4, Cpal and Srdbal genes are lowly expressed at 1 and 2 weeks, and highly expressed at 3 and 4 weeks transiently, and then decreased at 5 to 8 weeks. The results of Western blotting and immunohistochemical testing are consistent with the results of gPCR assay. The above results show that the verification results are consistent with the results of differentially expressed genes, indicating that the Slc38a4, Cpal and Srdbal genes are the marker genes of rat testicular progenitor Leydig cells, and the corresponding proteins are the marker proteins of rat testicular progenitor Leydig cells.
[0038]In view of the fact that rat testicular PLC marker protein has good specificity and sensitivity, the present disclosure provides use of the rat testicular progenitor
Leydig cell marker protein or the rat testicular progenitor
Leydig cell marker protein obtained by the screening method in identifying or screening rat testicular progenitor Leydig cells.
[0039]The rat testicular progenitor Leydig cell marker protein, the screening method and use thereof provided by the present disclosure will be described in detail below in conjunction with embodiments, but these embodiments should not be construed as limiting the protection scope of the present disclosure.
[0040] Example 1 [0041 ]A method for screening rat testicular progenitor Leydig cell marker protein
[0042]1. Reagents
[0043]4% paraformaldehyde, trizol, RIPA enhanced lysis buffer, Beyotime BCA kit, anhydrous ethanol, DEPC water, PBS solution, 10X loading buffer, 15% lower layer gel, neutral gum, sigma hard wax, reverse transcription kit from Promega (Madison, Wisconsin), 480 Green I Master from Roche (Basel,
Switzerland).
[0044] Sources of antibodies required for Western blotting (WB) :
[0045]1) Brand: Abcam, antibody name: Recombinant
Anti-Carboxypeptidase A antibody [EPR24384-69], Art. No.: {ab278044), applicable to IHC-Fr, mlHC, WB, IHC-P;
[0046]2) Brand: antibodies-online.com, Antibody name:
SLC38A4 antibody, Art. No.: (AA 381-430); SLC38A4 Reactivity:
Human, Mouse, Rat, Dog, Cow, Monkey; applicable to: WB, IHC,
IHC (p); Host: Rabbit, Polyclonalunconjugated;
[0047]3) Brand: Cell Signaling Technology, CYP17A1 (E6ATG)
Rabbit mAb #94004; applicable to:
[0048] Applicable to: WB, immunohistochemistry (frozen), co-immunoprecipitation; Species Reactivity: Human, Mouse,
Rat.
[0049] gPCR primer sequence:
[0050]S1c38ad F: CACCGTCTGCATCTTTCTCC (SEQ ID NO:4);
[0051]S1c38Bad R: TGGCGTTTGCTTTCGTCT (SEQ ID NO:5);
[0052] Cpal F: ATCCAGCGTATGATAGGTG(SEQ ID NO:6);
[0053]Cpal R: CCCCATTGATGTCAGAGTG(SEQ ID NO:7);
[0054]Srdbl F:CTCATGGGAGGCAACAGC (SEQ ID NO:8);
[0055]Srdbal R: CTCACCAGAGCGAAGCAG (SEQ ID NO:92);
[0056]D1kl F: AAGGACTGTCAGCACAAGG (SEQ ID NO:10);
[0057]D1kl R: ACAGAAGTTGCCCGAGAAG (SEQ ID NO:11);
[0058] Cypllal F: AAGTATCCGTGATGTGGGG (SEQ ID NO:12);
[0058]Cypllal R: TCATACAGTGTCGCCTTTTCT (SEQ ID NO:13);
[0060] CyplT7al F: TGGCTTTCCTGGTGCACAATC (SEQ ID NO:14);
[0061]Cypl7al R: TGAAAGTTGGTGTTCGGCTGAAG (SEQ ID NO:15);
[0062]RPS16 F18: AAGTCTTCGGACGCAAGAAA (SEQ ID NO:16);
[0063]RPS16 R18: TTGCCCAGAAGCAGAACAG (SEQ ID NO:17).
[0064]2. Laboratory animals
[0065]8D rats at 15 to 21 days of gestation, adult SD rats of 1 to 8 weeks old and male rats on the first day of birth are purchased from Zhejiang Vital River Laboratory Animal
Technology Co., Ltd. These animals are kept at 22°C with free access to drinking water and feeds. They are not subjected to any chemical or procedural treatment and are only used for isolation of LCs. The experimental procedures are approved by the Animal Care and Use Committee of Wenzhou Medical
University and are in accordance with the Guide for the Care and Use of Laboratory Animals of National Institutes of
Health.
[0066] 3. Treatment method. The flow chart is shown in FIG. 2. The treatment method specifically includes the following steps:
[0067]1) Treat male rats with ethylene dimesulfonate (EDS) to prepare a testicular Leydig cell development model, collect testicles from the development model 1 week and 3 weeks after treatment with EDS, isolate cells from leydig for single-cell sequencing, and screen differentially expressed genes in testicular progenitor Leydig cells; [00€8]2) Isolate testicles using SD male rats of 1 to 8 weeks old as objects, perform gPCR to verify the differentially expressed genes using testicular tissues as test materials, and carry out Western blotting and immunchistochemical testing to verify proteins corresponding to the differentially expressed genes;
[0069]3) Isolate testicles using rat embryos from SD pregnant rats at 15 to 21 days of gestation as objects, perform qPCR to verify the differentially expressed genes using testicular tissues as test materials, and carry out Western blotting and immunohistochemical testing to verify proteins corresponding to the differentially expressed genes;
[0070]4) Isolate testicles using rats on the first day of birth as objects, perform gPCR to verify the differentially expressed genes using testicular tissues as test materials, and carry out Western blotting and immunohistochemical testing to verify proteins corresponding to the differentially expressed genes.
[0071]4. Preparation of testicular tissue
[0072]After 5 hours of adaption, rats are euthanized by CO: asphyxiation and testicles are removed. The testicular capsules are peeled off, and the testicular tissues are lysed in RIPA lysis buffer and Trizol, respectively; at the same time, a part of them are fixed in 4% paraformaldehyde for 48 hours.
[0073]5. Preparation of paraffin sections - tissue fixation, dehydration and embedding
[0074]1) Collect testicles from the control group and the groups 1 week and 3 weeks after EDS injection; fix 3 testicles in 5 to 10 times the volume of 4% paraformaldehyde solution for 48 hours; cut 4-5 um of the fixed testicular specimens and place them in an embedding box;
[0075]2) Soak in pure water for 30 minutes at room temperature; [007€]3) Gradient dehydration with ethanol: dehydrate with 75% ethanol, 853 ethanol, 95% ethanol I and II for 1 hour and 20 minutes each, and with 100% ethanol I and II for 1 hour each; [0C77]4) Transparency: xylene I and II for 30 minutes each;
[0078]5) Waxing: Immerse in soft wax for 1 hour and 30 minutes, hard wax I for 1 hour, and hard wax II for 2 hours;
[0079]6) Embedding;
[0080]7) Sectioning: Cut into cross-section sections with 5 um in thickness, take 1 section for every 10 sections, and put the cut sections in the section box for later use.
[0081]6. Immunohistochemical staining
[0082] Perform immunohistochemical staining for the target protein by taking the CPAl protein detection method as an example; Place the paraffin sections in a 60°C oven for 2 hours (not more than 2 hours);
[0083] Dewaxing and rehydration: Perform dewaxing with xylene
I and II for 15-20 minutes each, and perform rehydration with 100% ethanol I, 100% ethanol II, 95% ethanol, 85% ethanol, 75% ethanol for 5 minutes each (in a descending gradient),
then ddH20 or pure water for 5 minutes, and then wash with
PBS twice, 5 minutes each time (ethanol can wash away xylene and facilitate the water washing and staining of sections).
Note: Dewaxing must be performed thoroughly, and the time for dewaxing with xylene can be appropriately extended, but cannot be shortened (washing with PBS can be carried out on a shaker at a speed of 100 rpm);
[0084]Antigen retrieval by high temperature: Place the enamel cylinder containing citrate retrieval solution in a pressure kettle containing water (slightly less water), boil until boiling (high temperature or boiling for sodium citrate), then place the slide rack in the citrate (sodium citrate reaching the white handle), cover the lid tightly, start timing for 3-4 minutes after spraying, and turn off the heat source (quickly remove the safety valve; keep safety, or use a stick to deflate, or cover with a wet towel to open); take out the sodium citrate solution, cool it naturally (cool it for half an hour, then put it in cold water to cool) to room temperature, and then soak in PBS for 10 minutes;
[0085]Blocking with endogenous peroxidase: Shake off the water on the section, drawa circle with a Pap pen, add dropwise peroxidase blocking agent, protect from light at room temperature for 15 minutes, and wash with PBS for 3 times, 5 minutes each time;
[0086] Serum blocking: After gently drying the slide, quickly add 10% normal serum blocking solution {10% donkey serum or 10% goat serum or 10% rabbit serum; 10% serum of different species from the primary antibody (100% serum diluted with
PBS) is required to prepare the blocking solution, 1% for primary antibody, 1% for secondary antibody), block at room temperature for 60 minutes. (for secondary antibody host serum, 1f the primary antibody is from goat species and the secondary antibody is rabbit anti-goat, the rabbit serum is used) ;
[0087]Add primary antibody: dilute with 1% serum, gently shake off the serum to fill the histochemical circle (quickly not allowing the tissues to dry), and add primary antibody
(note that the serum on the tissue must be shaken off when adding primary antibody, otherwise the added primary antibody will be easily mixed in the tissue), stay overnight at 4°C (12 hours) (operate in a wet box; add an appropriate amount of water to the wet box first; if the histochemical circle is lost, fill it, otherwise it will easily spread and evaporate to dryness);
[0088]Rewarming: put the wet box at room temperature or 37°C to rewarm for 30 minutes, then wash with PBS for 3 times, 5 minutes each time (or 4 minutes) to prevent detachment and make the antigen-antibody binding more stable. Washing with
PBS can be performed on a shaker at a speed of 100 rpm;
[0089] Enhancement solution: add enhancement solution at room temperature for 20 minutes, wash 3 times with PBS, 5 minutes each time;
[0080]Add secondary antibody: prepare secondary antibody with PBS or 1% goat serum or add the secondary antibody, incubate the secondary antibody at 37°C in a wet box (also shake off the liquid on the tissue when adding a secondary antibody) for 120 minutes, then wash 3 times with PBS, 5 minutes each time;
[0091]1DAB color development: Prepare and use the color development agent (20:1) immediately prior to use, protected from light; shake and centrifuge, place on ice, protected from light; spindry the slice, add dropwise the tissue with 100-200 ul gun, and observe the time when the Leydig turns brown under the microscope (color change in 3 minutes for CYP17Al); put it in clear water after it turns slightly brown, and then rinse with tap water for 15 minutes;
[0092]Staining with hematoxylin differentiation solution:
Prepare a basin of clear water in advance, shake the slides to dryness, add dropwise hematoxylin for 10-30 seconds, put itinclearwater andrinse for 15minutes; adddifferentiation solution for 10-30 seconds, put into clear water and rinse for 15 minutes; (Note: it is not recommended to draw hematoxylin fully as it is easy to stain the gun body).
[00983]Dehydration: Treat with ethanol (75%, 85%, 95%, 95%,
100%, 100%) each for 3 minutes successively;
[0094] Transparency: Treat with xylene I and xylene II for 15-20 minutes separately, dry the neutral resin slides in the air for 10 minutes, seal, and photograph 15 minutes later.
[0085]7. RNA extraction and quantitative polymerase chain reaction (gPCR)
[0086] Total RNA is extracted using the Mini Kit (Qiagen,
Germany) according to the instructions, and cDNA is synthesized, and gene detection is performed by QPCR. All major genes related to T synthesis and metabolism, including
Slc38a4, Cypllal, Cypl7al, Cpal, Srdbal, and Dlkl, are analyzed. The mRNA level of ribosomal protein S16 (Rpslé) is used as an internal standard, and the Ct value of the target gene is normalized according to the Ct value of Rpsl16.
[0097]8. Western blotting (WB)
[0088] The total cell protein is extracted using RIPA lysis buffer (Beyotime, China), and the protein concentration is determined using the Enhanced BCA Protein Concentration Assay
Kit (Beyotime, China). Sample protein (700 ng) is separated by 10% SDS-PAGE and transferred to a polyvinylidene fluoride membrane. The proteins to be analyzed include CPAl, CYP17Al, and SLC38A4. Using GADPH as the internal standard protein and based on its gray value, the gray value of each target protein is quantitatively analyzed.
[0099]9. Data analysis
[0100] The data are expressed as the mean + SE (standard error) of 3-4 independent experiments. The t-test is conducted to verify whether there is a significant difference between the two groups of experiments. ANOVA is used for multi-group comparison, and the SNK test is conducted to determine the significant differences among groups. *P<0.05 is considered statistically significant difference.
[0101]Results
[0102]1. SRD5A1, CPAl, SLC38A4, and DLK1 are specifically expressed in testicle LCs in rats
[0103]In order to find a cell surface marker protein that can be used to isolate adult PLCs from rodents, the single-cell sequencing is perform, and SRD5A1, CPAl, SLC38A4, and DLK1 are found to be possibly appropriate proteins. The scRNA-seq analysis report shows that SRD5SA1, CPAl, SLC38A4, and DLK1 are the best markers that are specifically expressed in PLCs among various types of leydig cells.
[0104] Generally, single-cell sequencing data display the relationship among cells in 2D tSNE. In FIG. 3, each point represents a cell, and the position of cells is determined by its transcriptome characteristics. Similar cells are aggregated together to become small islands. By sequencing rat Leydig cells after treatment with EDS, the SLC (blue),
PLC (green), and ALC (red) are identified. The single gene distribution diagram on the right shows that Col4a4 is only expressed in SLC, Clec3b is expressed in SLC and PLC, and
Srdbal is only expressed in PLC. Cypllal is expressed in PLC and ALC, while Pah is only expressed in ALC. In addition, a variety of other genes that are only expressed in PLC are also identified.
[0105]The results of PLC cells expressing various marker genes are shown in FIG. 4. A systematic display of PLC cells expressing other cell genes is made. SLC genes are lowly expressed in PLCs, and most of them cannot be detected, but
ALC testosterone synthesis genes are highly expressed.
However, detoxification genes are lowly expressed in ALC cells. In addition, low levels of cell division genes are also expressed in PLC cells.
[0106] The RT-gPCR results are shown in FIG. 5. By using Cypllal and Cypl7al as controls, the genes Cpal, Srdbal and Dlkl are highly expressed in embryonic Leydig cells at 19 days of gestation and in PLCs during 21 and 28 days of puberty development, but are lowly expressed in testicles of newborn and adult rats. The Slc38a4 gene expression is statistically different between GD18-21 and the day of birth, and is highly expressed 3 and 4 weeks after birth, with no statistical difference comparedwith the group on the day of birth; however, the trend between 3 and 4 weeks is consistent with the WB results. The reason is possibly the decrease in the proportion of Leydig cells in the total testicular cells after birth and the dilution of the total amount of genes. The overall trend is consistent with the WB results.
[0107] The Western blotting results are shown in FIG. 6. FIG. 6 is statistically analyzed to obtain FIG. 7. *P<0.05 is considered statistically significant difference. The statistical analysis with Birth (0 days after birth) is conducted in all groups, and the data are expressed as mean t SD (standard deviation) (n=3). High expression is observed at GD19, 21 and 28 days after birth; and the data are expressed as mean + SE (standard error) of 3-4 independent experiments.
The t-test is conducted to verify whether there is a significant difference between the two groups of experiments.
ANOVA is used for multi-group comparison, and the SNK test is conducted to determine the significant differences among groups. As shown from the above results, using GADPH as an internal standard, the expressions of the testosterone synthesis protein and PLC-specific proteins (SRD5SA1, CPA1,
SLC38A4, and DLK1) are detected by WB.
[0108]In order to confirm that CPAl protein is specifically expressed by PLCs, CPAl immunchistochemical staining is performed on embryonic testicular sections and adult rat testicular sections (FIG. 8). CPAl is highly expressed in the
Leydig at 16-18 days of gestation, highly expressed at 3 and 4 weeks after birth, and decreased after birth and during maturity. None of them is expressed by any vas deferens cells.
[0109]According to the results of above embodiments, the four proteins (SLC38A4, CPAl, DLK1 and SRD5A1) appear specifically in the PLC stage during the development of ALC, and can be used as marker proteins for PLC cells.
[0110] The foregoing description merely describes the preferred embodiments of the present disclosure. It should be noted that for those of ordinary skill in the art, several improvements and modifications can be made without departing from the principle of the present disclosure, and these improvements and modifications shall also fall within the scope of protection of the present disclosure.
HKJP20240402338 CN 202311588171.6 2023-11-25 The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University RAT TESTICULAR
PROGENITOR LEYDIG CELL MARKER PROTEIN, SCREENING METHOD AND USE
THEREOF 17 547 AA PAT source 1..547 mol_type protein note Amino acid sequence of SLC38A4 organism unidentified
MDPIELRSVNIEPYEDSCSVDSIQSCYTGMGNSEKGAMDSQFANEDAESQKFLTNGFLGKKT
LTDYADEHHPGTTSFGMSSFNLSNAIMGSGILGLSYAMANTGIVLFVIMLLTVAILSLYSVHLLL
KTAKEGGSLIYEKLGEKAFGWPGKIGAFISITMQNIGAMSSYLFIIKYELPEVIRVFMGLEENTG
EWYLNGNYLVLFVSVGIILPLSLLKNLGYLGYTSGFSLTCMVFFVSVVIYKKFQIPCPLPVLDHN
NGNLTFNNTLPMHVIMLPNNSESTGMNFMVDYTHRDPEGLDEKPAAGPLHGSGVEYEAHSG
DKCQPKYFVFNSRTAYAIPILAFAFVCHPEVLPIYSELKDRSRRKMQTVSNISITGMLVMYLLAA
LFGYLSFYGEVEDELLHAYSKVYTFDTALLMVRLAVLVAVTLTVPIVLFPIRTSVITLLFPRRPFS
WVKHFGIAATIIALNNVLVILVPTIKYIFGFIGASSATMLIFILPAAFYLKLVKKEPLRSPQKIGALV
FLVTGIIFMMGSMALIIIDWIYNPPNPDHH 419 AA PAT source 1..419 mol_type protein note Amino acid sequence of CPA1 organism unidentified
MKRLLILSLLLEAVCGNENFVGHQVLRISAADEAQVQKVKELEDLEHLQLDFWRDAARAGIPI
DVRVPFPSIQSVKAFLEYHGISYEIMIEDVQLLLDEEKQQMSAFQARALSTDSFNYATYHTLDE
IYEFMDLLVAEHPQLVSKIQIGNTFEGRPIHVLKFSTGGTNRPAIWIDTGIHSREWVTQASGV
WFAKKITKDYGQDPTFTAVLDNMDIFLEIVTNPDGFAYTHKTNRMWRKTRSHTQGSLCVGVD
PNRNWDAGFGMAGASSNPCSETYRGKFPNSEVEVKSIVDFVTSHGNIKAFISIHSYSQLLLYP
YGYTSEPAPDQAELDQLAKSAVTALTSLHGTKFKYGSIIDTIYQASGSTIDWTYSQGIKYSFTF
ELRDTGLRGFLLPASQIIPTAEETWLALLTIMDHTVKHPY 255 AA PAT source 1..255 mol_type protein note Amino acid sequence of SRD5A organism unidentified
MELDELCLLDMLVYLEGFMAFVSIVGLRSVGSPYGRYSPQWPGIRVPARPAWFIQELPSMAWP
LYEYIRPAAARLGNLPNRVLLAMFLIHYVQRTLVFPVLIRGGKPTLLVTFVLAFLFCTFNGYVQSR
YLSQFAVYAEDWVTHPCFLTGFALWLVGMVINIHSDHILRNLRKPGETGYKIPRGGLFEYVSAA
NYFGELVEWCGFALASWSLQGVVFALFTLSTLLTRAKQHHQWYHEKFEDYPKSRKILIPFVL
DNA PAT source 1..20 mol_type other DNA note Primer Slc38a4 F organism synthetic construct caccgtctgcatctttctcc 18 DNA PAT source 1..18 mol_type other
DNA note Primer Slc38a4 R organism synthetic construct tggcgtttgctttcgtct 19 DNA
PAT source 1..19 mol_type other DNA note Primer Cpal F organism synthetic construct atccagcgtatgataggtg 19 DNA PAT source 1..19 mol_type other DNA note
Primer Cpal R organism synthetic construct ccccattgatgtcagagtg 18 DNA PAT source 1..18 mol_type other DNA note Primer Srd51 F organism synthetic construct ctcatgggaggcaacagc 18 DNA PAT source 1..18 mol_type other DNA note Primer
Srd5al R organism synthetic construct ctcaccagagcgaagcag 19 DNA PAT source 1..19 mol_type other DNA note Primer DIk1l F organism synthetic construct aaggactgtcagcacaagg 19 DNA PAT source 1..19 mol_type other DNA note Primer
DIk1 R organism synthetic construct acagaagttgcccgagaag 19 DNA PAT source 1..19 mol_type other DNA note Primer Cypllal F organism synthetic construct aagtatccgtgatgtgggg 21 DNA PAT source 1..21 mol_type other DNA note Primer
Cypllal R organism synthetic construct tcatacagtgtcgccttttct 21 DNA PAT source 1..21 mol_type other DNA note Primer Cyp1l7al F organism synthetic construct tggctttcctggtgcacaatc 23 DNA PAT source 1..23 mol_type other DNA note Primer
Cyp17al R organism synthetic construct tgaaagttggtgttcggctgaag 20 DNA PAT source 1..20 mol_type other DNA note Primer RPS16 F18 organism synthetic construct aagtcttcggacgcaagaaa 19 DNA PAT source 1..19 mol_type other DNA note
Primer RPS16 R18 organism synthetic construct ttgcccagaagcagaacag
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5679521A (en) * | 1990-04-30 | 1997-10-21 | Board Of Regents, The University Of Texas System | Steroid 5A-reductases |
| US20020082191A1 (en) * | 2000-12-08 | 2002-06-27 | Karl Guegler | Isolated human transporter proteins, nucleic acid molecules encoding human transporter proteins, and uses thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5679521A (en) * | 1990-04-30 | 1997-10-21 | Board Of Regents, The University Of Texas System | Steroid 5A-reductases |
| US20020082191A1 (en) * | 2000-12-08 | 2002-06-27 | Karl Guegler | Isolated human transporter proteins, nucleic acid molecules encoding human transporter proteins, and uses thereof |
Non-Patent Citations (5)
| Title |
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| ANONYMOUS: "CAS2:483187388_1", 29 January 2003 (2003-01-29), XP093268756, Retrieved from the Internet <URL:http://ibis.internal.epo.org/exam/dbfetch.jsp?id=CAS2:483187388_1> * |
| GUAN XIAOJU ET AL: "Identification of Rat Testicular Leydig Precursor Cells by Single-Cell-RNA-Sequence Analysis", FRONTIERS IN CELL AND DEVELOPMENTAL BIOLOGY, vol. 10, 15 February 2022 (2022-02-15), XP093268663, ISSN: 2296-634X, DOI: 10.3389/fcell.2022.805249 * |
| LI L ET AL: "Regulation of development of rat stem and progenitor Leydig cells by activin", ANDROLOGY, SCRIPTOR PUBLISHER APS, HOBOKEN, USA, vol. 5, no. 1, 27 September 2016 (2016-09-27), pages 125 - 132, XP072453011, ISSN: 2047-2919, DOI: 10.1111/ANDR.12253 * |
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