Application of mitochondrial formation factor in diagnosis or treatment of male fertility defect

Abstract

The invention provides a kit for diagnosing fertility defects such as infertility in mammals with different sexes. The kit comprises a reagent for detecting MORN2 of a tested individual. The invention also provides kits and instruments for diagnosing or predicting fertility defects, such as infertility disorders, in mammalian individuals by detecting MORN2. Applicants thus provide a method of diagnosing fertility defects such as infertility in mammals, including humans, further facilitating the development of new therapeutic methods for male infertility individuals affected by gene mutations.

Description

Technical Field

[0001] The present invention relates to the field of diseases and diagnosis. Specifically, the present invention relates to methods and kits for diagnosing or treating fertility defects in mammals, especially humans. Background Art

[0002] Fertilization is the union of female (oocyte) and male (sperm) gametes, and the formation of mature sperm is the result of a series of processes, roughly referred to as mitosis, meiosis, and spermatogenesis. Spermatogenesis is a transformation stage in which round sperm undergoes acrosome formation, nuclear condensation, flagella development, mitochondrial sheath formation, and removal of unnecessary cytoplasmic components to eventually become mature sperm. Spermatozoa have a haploid head, neck, and flagellum. The flagellum is divided into three regions (midsection, principal segment, and tail segment). The flagellum is the main power unit, providing the required force for sperm transport to the oocyte.

[0003] The flagellar middle piece has a structure called the mitochondrial sheath, which is tightly wrapped around the axoneme. The formation of the mitochondrial sheath occurs in the late spermatogenesis, during which spherical mitochondria are recruited from the cytoplasm and arranged around the flagellum (the "arrangement step"). The mitochondria then elongate outward to form a crescent shape (the "linking step"). Subsequently, the crescent-shaped mitochondria are further elongated and tightly wrapped around the axoneme in a double helix structure (the "compaction step"). Although the total number of mitochondria decreases as the cytoplasm decreases, the compact mitochondrial sheath enables the condensed mitochondria to have high metabolic efficiency. The ultrastructure and formation process of the mitochondrial sheath have been described in previous studies, and it is known that knocking out several biomolecules (such as ARMC12, SPATA33, and VDAC3) will lead to abnormal mitochondrial sheath formation and infertility, but the key pathways of mitochondrial sheath formation and the detailed molecular mechanisms of related defects remain largely unknown.

[0004] Mitochondria have double membranes and energy metabolism functions, producing adenosine triphosphate (ATP) through oxidative phosphorylation (OXPHOS) to power sperm differentiation and motility. The inner membrane of mitochondria, the cristae, is highly folded, increasing its surface area, which greatly upregulates the storage of OXPHOX complexes and ATP synthase in the cristae. Previous studies have reported that the levels of proteins related to mitochondrial OXPHOS, TCA cycle, and pyruvate metabolism in sperm from patients with asthenozoospermia are abnormally low, and another study has shown that reduced mitochondrial respiratory activity can lead to poor sperm motility and asthenozoospermia.

[0005] In addition to ATP, mitochondria can also produce reactive oxygen species (ROS) through OXPHOX. ROS are reactive oxygen-derived molecules with one or more unbound electrons. At appropriate concentrations, ROS are essential for the acrosome reaction, sperm-oocyte fusion and fertilization, while excessive ROS production can mediate sperm damage and male infertility. During the oxygenation process, electrons are transferred from donors to acceptors. Through this series of redox reactions, the mitochondrial electron transport chain forms an electrochemical gradient and generates mitochondrial membrane potential (MMP). Normal MMP is essential for ATP generation, and monitoring MMP can be used to assess mitochondrial function. It has been reported that low MMP is one of the main causes of poor sperm motility and severe asthenozoospermia.

[0006] Previous studies have predicted that MORN2 may be involved in the dynamic regulation of acrosome biogenesis in the late stage of spermatogenesis. However, the specific role of MORN2 in spermatogenesis or reproduction is still unclear.

[0007] The field also needs to have a better understanding of the male spermatogenesis process in mammals, so as to detect and predict reproductive disorders such as infertility. Summary of the invention

[0008] The inventors discovered for the first time that Morn2 is not only involved in the formation of the mitochondrial sheath, but also in the energy metabolism of sperm mitochondria. The research of this application revealed the potential pathogenic gene of asthenozoospermia and suggested a potential target for the development of male contraceptive technology. The inventors of this application found that Morn2 regulates the morphology and energy metabolism of mitochondria and is necessary for male fertility; at the same time, it was found that Morn2 may have an effect on female fertility, but it is not essential. Morn2- / -Male mouse infertility, Morn2- / -Sperm exhibits an abnormal phenotype of inverted head and extremely reduced motility. MORN2 has a potential interaction with SK2 in the testis, affecting electron transport in germ cells, thereby affecting sperm membrane potential and ultimately regulating sperm motility. Thus, the inventors provide a method for diagnosing or treating fertility defects such as infertility in mammals, especially humans.

[0009] In one aspect of the present invention, a method for diagnosing fertility defects such as infertility in mammals of different sexes by detecting U MORN2 in the test individual is provided. The method may further include the step of detecting the sex of the test individual.

[0010] In one aspect of the present invention, a kit for diagnosing fertility defects such as infertility in mammals of different sexes is provided, which comprises a reagent for detecting MORN2 in a test subject. In the present invention, the kit also comprises a reagent for detecting the sex of the test subject, in particular, whether the test subject is male.

[0011] The present invention thus provides the use of a reagent for detecting MORN2 in a test subject in the preparation of a kit for diagnosing fertility defects such as infertility in mammals of different sexes. In the present invention, the kit also includes a reagent for detecting the sex of the test subject.

[0012] In the present invention, infertility disorders include but are not limited to teratozoospermia, azoospermia, oligozoospermia, asthenozoospermia, asthenozoospermia and the like.

[0013] In one aspect of the present invention, the infertility disorder is asthenozoospermia or asthenozoospermia. The inventors of the present application have found that Morn2 regulates mitochondrial morphology and energy metabolism and is essential for male fertility. / -Male mouse infertility, Morn2- / - Spermatozoa showed an abnormal phenotype of inverted head and extremely reduced motility. Abnormal formation of mitochondrial sheath detected by transmission electron microscopy and scanning electron microscopy, as well as abnormal energy metabolism characterized by low MMP, high ROS and significantly reduced ATP concentration. The in vitro fertilization experiment of the embodiment showed that Morn2- / -Sperm can penetrate the ZP and fuse with the oocyte; however, fertilization has not been observed in vivo. This is consistent with Morn2- / -Related to low sperm motility. Morn2- / -Sperm cannot pass through the female reproductive tract to reach the ampulla of the fallopian tube to combine with the egg, resulting in male infertility.

[0014] In the present invention, the mammal can be any mammal, including and not limited to rodents, canines, felines, equines, ovines, bovines, porcines and primates. The mammal typically includes humans.

[0015] MORN2, or membrane occupancy and recognition junctional motif protein 2. The Ensembl database (http: / / asia.ensembl.org / index.html) shows that MORN2 is evolutionarily conserved in animals such as humans, chimpanzees, dogs, cows, rats, chickens, zebrafish, and frogs. The Gene ID of the human Morn2 gene encoding MORN2 is 729967; the Gene ID of the mouse Morn2 gene is 378462. In mice, MORN2 is enriched in the testes, and its initial expression coincides with the occurrence of the first wave of meiosis; Morn2 is located on chromosome 17E3, with a total length of approximately 7kb, containing 6690 base pairs of nucleotides and corresponding 79 amino acids of the mouse open reading frame. MORN2 has been shown to play a role in planarian LC3-associated phagocytosis and resistance to bacterial infection.

[0016] In the application (including methods and related kits) provided by the present invention for diagnosing fertility defects or infertility in mammals of different sexes by detecting MORN2 of the test individual, whether the MORN2 of the test individual is abnormal is detected, which includes comparing the activity of the subject's MORN2 gene or its regulatory factor, or the expression of MORN2 protein, or comparing with the situation of the gene or protein in normal people in the general or specific group. If there is a significant change, it is judged that an abnormality exists.

[0017] In one aspect of the present invention, the method and kit for diagnosing fertility defects provided by the present invention include steps and reagents for detecting the genes of each component of the aforementioned complex, such as detecting the Morn2 gene encoding MORN2.

[0018] The method for detecting genes used in the present invention is a method commonly used for detecting genes in the art and is not particularly limited. For example, mass spectrometry, microarray, sequencing, and detection methods using base sequence amplification methods such as PCR (polymerase chain reaction) can be cited.

[0019] In addition, the detection of PCR products obtained when PCR is performed using primers specific to each gene can be performed by any method commonly used to detect and quantify PCR products. For example, it can be detected by electrophoresis, by real-time PCR using fluorescent intercalators such as SYBR Green, or by single molecule fluorescence analysis.

[0020] The polynucleotide that can be used as the primer or probe for detecting gene is not particularly limited as long as it can be a polynucleotide that hybridizes with the partial region comprising the gene or its complementary strand. The design of polynucleotide can be carried out by any one of the well-known methods in the technical field. For example, it can be designed simply by utilizing known genome sequence data and universal primer design tool. As this primer design tool, for example, Primer3 that can be utilized on the network is arranged. In addition, known genome sequence data can be obtained in NCBI as an international base sequence database usually.

[0021] In one aspect of the present invention, the method and kit for diagnosing fertility defects provided by the present invention include a step of detecting MORN2 protein, such as a step of detecting MORN2 protein, including detecting the expression of the protein (including whether it exists, and whether the expression is reduced relative to normal or standard) or its activity.

[0022] In another aspect of the present invention, the method and kit include steps and reagents for immunoassaying the expression of each component protein in the aforementioned complex. For example, the expression of the target protein is detected by immunofluorescence or Western blotting with an antibody that specifically recognizes the protein. For another example, the expression of the target protein is detected by detecting the presence or amount of mRNA of the protein, such as by detecting the amount of mRNA encoding the target protein in the sample by RT-PCR.

[0023] In yet another aspect of the present invention, the method and kit include steps and reagents for determining the activity of MORN2 protein.

[0024] The present invention also provides an apparatus (including a gene chip) for diagnosing or predicting fertility defects such as infertility in mammalian individuals.

[0025] The method and kit provided by the present invention are based on the discovery that the effect of MORN2 on the fertility of mammals is gender-dependent, so the diagnosis and prediction made by the method or kit provided by the present invention are different for mammals of different genders. Therefore, in the method provided by the present invention, it is also included to detect the gender of the subject. In the kit provided by the present invention, it is also included to detect the gender of the subject. Methods and reagents for detecting the gender of the subject are well known in the art, for example, including detecting the gender-specific proteins or genes of the subject, such as specific nucleic acid sequences on sex chromosomes, etc.

[0026] In this article, protein symbols are not italicized and are all capitalized; gene symbols are italicized. For example, MORN2 is a protein, and the gene encoding the protein is written as Morn2. Sometimes in this article, protein symbols are not all capitalized. For example, sometimes "Morn2" in this article means MORN2 protein. Sometimes in this article, gene symbols are not italicized. For example, sometimes "Morn2" or "MORN2 gene" in this article means the gene Morn2 encoding MORN2 protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 showed that Morn2 gene knockout resulted in male infertility.

[0028] (A)Morn2 - / - Overview of mouse knockout strategies.

[0029] (B) From Morn2 - / - 、Morn2 + / + and Morn2 + / - Genotyping analysis of lysates prepared from mouse tails showed successful knockout of Morn2.

[0030] (C)Morn2 + / - and Morn2 - / - Representative photographs of mouse testes.

[0031] (D) Adult Morn2 + / - and Morn2 - / - Mouse body weight.

[0032] (E) Adult Morn2 + / - and Morn2 - / - Mouse testis weight.

[0033] (F)Morn2 + / - and Morn2 - / - Testis weight / body weight ratio of mice.

[0034] (G)Morn2 + / - and Morn2 - / - Number of pups per litter of male mice. + / - In mice, 10 litters were produced, with 7.40 ± 1.90 pups per litter. - / - In mice, the litter size was 0. Student's t-test was performed, and the data are presented as the mean ± SEM of 15 independent experiments. ns: P>0.05, ****: P<0.0001.

[0035] Figure 2 Show Morn2 - / - Mouse spermatozoa exhibit inverted heads.

[0036] (A)Morn2 + / - and Morn2 - / - Mouse testis stained with hematoxylin. The scale bar is 20 μm.

[0037] (B)Morn2 + / - and Morn2 - / - Mouse epididymis stained with hematoxylin. Scale bar is 50 μm.

[0038] (C)Morn2 + / - and Morn2 - / - Mouse epididymal sperm smear stained with hematoxylin and eosin. Scale bar is 20 μm.

[0039] (D)Morn2 + / - and Morn2 - / - Percentage of epididymal sperm with abnormal phenotype in mice.

[0040] (E)Morn2 + / - and Morn2 - / - The proportion of inverted sperm heads in mouse epididymis. Student's t-test was performed, and the data are presented as the mean ± SEM of three independent experiments. ***: P < 0.001.

[0041] (F)Morn2 + / - and Morn2 - / - PAS staining of different stages of mouse seminiferous tubules. (Pl, preleptotene spermatocyte; L, leptotene spermatocyte; Z, zygotene; P, pachytene spermatocyte; RS, round spermatid; ES, elongated spermatid; M, meiotic metaphase. Scale bar is 5 μm.)

[0042] Figure 3 Show Morn2 - / - Sperm motility decreased significantly.

[0043] (A) TUNEL staining of testicular sections showed that Morn2 - / - Morn2 + / - There are more apoptotic cells in mice, and most of the apoptotic cells are round and elongated sperm. Arrows indicate apoptotic cells. The scale bar is 70 μm.

[0044] (BD)Morn2 + / - and Morn2 - / - Average path velocity (VAP) (B), forward velocity (VSL) (C) and trajectory velocity (VCL) (D) of mouse sperm (n=3). ****p<0.0001.

[0045] (E)Morn2 + / - and Morn2 - / -Percentage of motile sperm in mice. ****p<0.0001.

[0046] (F)Morn2 + / - and Morn2 - / - The percentage of fast-moving sperm in mice. ****p<0.0001.

[0047] (G)Morn2 + / - and Morn2 - / - In vitro fertilization rate of mice. *p<0.05.

[0048] (H)Morn2 + / - and Morn2 - / - Fertilization rate in mice. Student's t-test was performed, and the data are presented as the mean ± SEM of three independent experiments. ****p<0.0001, ***p<0.001, **p<0.01.

[0049] Figure 4 It was shown that Morn2 gene knockout resulted in abnormal sperm mitochondrial morphology.

[0050] (A)Morn2 + / - and Morn2 - / - Transmission electron microscopy (TEM) analysis of mouse epididymal sperm. Morn2 - / - The mitochondria in the middle of the sperm flagellum are enlarged and swollen, and the outer dense fibers are disordered and proliferated. Red arrows: outer dense fibers; white arrows: mitochondrial sheaths. The scale bar is as shown in the figure.

[0051] (B)Morn2 + / - and Morn2 - / - Scanning electron microscopy (SEM) analysis of mouse epididymal sperm. Scale bars are indicated in the figure.

[0052] (C) Transmission electron microscopy showed that Morn2 - / - Mouse sperm axons are disorganized. Scale bars are indicated in the figure.

[0053] (D) Transmission electron microscopy showed that Morn2 - / - The outer layer of dense fibers in the middle section of mouse axons proliferates, and the arrangement of mitochondrial sheaths is severely disrupted. Red (dark in grayscale) arrows: outer layer of dense fibers; blue (medium dark in grayscale) arrows: radial spokes; yellow (light in grayscale) arrows: mitochondrial sheaths; 1: microtubule dyad; 2: central microtubule.

[0054] Figure 5 Show Morn2 - / - Abnormal sperm energy metabolism in mice.

[0055] (A) Using JC-1 as a marker to detect adult Morn2 + / -and Morn2 - / - Mitochondrial membrane potential of mouse epididymal cauda sperm. Red (dark in grayscale) fluorescent signal indicates high potential, and green (light in grayscale) fluorescent signal indicates low potential. The scale bar is 30 μm.

[0056] (B)Morn2 - / - Low MMP signaling in mice is higher than in Morn2 + / - Mice. **p<0.01.

[0057] (C)Morn2 + / - and Morn2 - / - Analysis of ROS levels in sperm from the cauda epididymis of adult mice.

[0058] (D)Morn2 - / - ROS levels in mice were higher than in Morn2 + / - Mice. **p<0.01.

[0059] (E)Morn2 + / - and Morn2 - / - Analysis of ATP levels in spermatozoa of the cauda epididymis of adult mice. N = 3, **p < 0.01. Student's t-test was performed and the data are presented as the mean ± SEM of three independent experiments. DETAILED DESCRIPTION

[0060] The essential contents and beneficial effects of the present invention will be further described below in conjunction with embodiments, which are only used to illustrate the present invention rather than to limit the present invention.

[0061] Example 1 Experimental methods and reagents

[0062] Experimental animals

[0063] The Morn2 gene (Transcript: ENSMUST00000061703) is located on mouse chromosome 17 and contains 5 exons. Exons 4 to 5 were selected as targets and the Morn2- / -Mouse model. Briefly, Cas9 and gRNA were co-injected into fertilized eggs to generate a targeted line with a 1841 bp base deletion. Genotyping was performed by polymerase chain reaction (PCR), and DNA sequencing analysis was performed. All mice were housed in a specific pathogen-free (SPF) animal facility. All animal experiments were performed in accordance with protocols approved by the Animal Use Committee of Shandong University School of Medicine.

[0064] RT-PCR

[0065] Total RNA was isolated from multiple adult tissues (including liver, spleen, lung, kidney, uterus, ovary and testis) of wild-type C57BL / 6 mice and testis of 8-56-day-old mice using FastPure Cell / Tissue Total RNA Isolation Kit V2 (Vazyme, RC112-01). All cDNAs were prepared using HiScript Q RT SuperMix for qPCR (Vazyma, R323-01) according to the manufacturer's instructions. RT-PCR experiments were first denatured at 95°C for 5 min, then denatured at 95°C for 30 s, retarded at 60°C for 30 s, extended at 72°C for 1 min, and finally extended at 72°C for 10 min using a T100 thermal cycler (BioRad) for 35 cycles.

[0066] Primer sequences for Morn2:

[0067] Forward:5-CGTCTCCATCAACTTCGTC-3;

[0068] Reverse:5-CTGTCCCGTTTTCTCACA-3.

[0069] Gapdh was amplified as a housekeeping gene with the following primers:

[0070] Forward:5-GCCTTCTCCA TGGTGGTGAA-3;

[0071] Reverse:5-GCACAGTCAAGGCCGAGAAT-3.

[0072] Tissue collection and histological analysis

[0073] Immediately after euthanasia, mouse testes, epididymis, and ovaries were dissected and fixed with 4% (mass / volume) paraformaldehyde (PFA) (Solarbio, Beijing, China, P1110) or Bouin solution (Sigma, HT10132) for 24 hours, dehydrated with 70% (volume / volume) ethanol, and embedded in paraffin. The tissue was cut into 5 μm thick sections and mounted on glass slides. After dewaxing and rehydration, the sections were stained with hematoxylin (H), hematoxylin-eosin (H&E), or PAS for histological analysis. The sections were also used for TUNEL staining and the manufacturer's instructions (keyGENBioTECH, #KGA7072) were followed.

[0074] Sperm collection and sperm immunofluorescence staining

[0075] Morn2+ cells were dissected immediately after euthanasia / - and Morn2- / - Mouse cauda epididymis. Sperm was squeezed out of the cauda epididymis, placed in 1 ml phosphate buffered saline (PBS), released for 20 min at 37°C, 5% CO2 gas environment, and then heated at 60°C for 10 min to inactivate the sperm. 20 μl of the sample was spread on a slide, air-dried, and stored at -80°C for further morphological analysis and immunofluorescence experiments. After fixation in 4% PFA for 15 minutes, the samples were washed 3 times with PBS and rehydrated, and stained with hematoxylin and eosin (HE) for histological analysis.

[0076] For immunofluorescence, slides were fixed in 4% PFA for 15 min at room temperature and washed three times in 1x PBS and permeabilized with 0.1% Triton for 10 min. Subsequently, slides were blocked with 5% BSA in a humidified chamber at room temperature for at least 30 min. Slides were then incubated with primary antibodies in a humidified chamber at 4°C overnight. Afterwards, they were washed three times with PBS and incubated with secondary antibodies for 1 h at room temperature. They were then rinsed three times with PBS and finally stained with DAPI (Abcam, ab104139) and imaged on an SP8 microscope (Leica). The primary and secondary antibodies used and their dilutions are listed in Table S1.

[0077] Testis immunofluorescence staining

[0078] Paraffin-embedded sections were prepared as described previously. Sections were dewaxed, rehydrated, and then antigen retrieved in boiling water for 15 min using 10 mM sodium citrate buffer (pH 6.0) (Proteintech, PR30001), cooled at room temperature, and then treated with PBS containing 0.1% Triton X-100.

[0079] Subsequent washing, blocking, and antibody incubations were consistent with the previously described protocol for fluorescent staining of sperm smears.

[0080] CASA analysis of sperm motility

[0081] At least three Morn2+ / - and Morn2- / -Mice sperm samples were analyzed for sperm motility assessment. Ten μl of suspended sperm from each sample were placed in an 80 mm deep glass cell chamber (Hamilton Thorne, 80 micron 2X-CEI) and captured using a CCD camera. Computer-associated semen analysis (CASA) was performed on more than 200 spermatozoa using the Animal Motility System (Hamilton Thorne, Beverly, MA, USA) to assess motility parameters, including total motile sperm, fast forward motile sperm, average path velocity (VAP), forward velocity (VSL), and trajectory velocity (VCL). Semen analysis was repeated at least three times.

[0082] In vivo / in vitro fertilization experiments

[0083] Oocytes were collected from 5-week-old postovulatory female WT and placed in mHTF medium covered with paraffin oil. / - and Morn2- / - Mouse (12-16 weeks old) epididymal cauda sperm were incubated with TYH drops for 30 min at 37°C in a 5% CO2 atmosphere. The eggs were then incubated with sperm for 6 h, and the fertilization rate was determined by counting the number of two-cell embryos at 24 h.

[0084] For in vivo fertilization, male individuals (WT and KO) were placed with superovulated female WT and the mating plug was detected at 9 a.m. the next day. 36 h after mating, the oviduct of the female mouse was opened, the contents of the ampulla of the oviduct were flushed with M2 medium (Sigma), and the number of two-cell embryos was counted to determine the fertilization rate.

[0085] Transmission electron microscopy (TEM)

[0086] Morn2+ cells were dissected immediately after euthanasia / - and Morn2- / - Mouse epicaudal testis (n=3), spermatozoa were squeezed out from epicaudal testis.

[0087] After washing by centrifugation, the samples were suspended in 2.5% glutaraldehyde, protected from light, and fixed overnight at 4°C. They were then immersed in 1% OsO4 for 1 h at 4°C in the dark and washed 4 times with distilled water. Subsequently, the samples were dehydrated through a graded acetone series and embedded in resin (low viscosity embedding medium Spurr's Kit, EMS, 14300) for staining. Finally, the samples were cut on an ultramicrotome, and ultrathin sections of the samples were double-stained with uranyl acetate and lead citrate, and imaged and analyzed using a transmission electron microscope (TEM) (JEM-1400, JEOL, Tokyo, Japan).

[0088] Transmission electron microscopy (SEM)

[0089] The sampling, fixing and washing methods of the samples were consistent with the above transmission electron microscopy. After washing, the samples were cold dehydrated with 50%, 70%, 95%, and 100% ethanol, respectively. Subsequently, the samples were dried at 55°C using a Tousimis autosamri-810 critical point dryer, mounted on sample stubs, and sealed with palladium. Finally, the samples were observed and analyzed using a FEI Quanta ESEM 200 scanning electron microscope.

[0090] Sperm membrane potential (MMP) assay

[0091] The protocol followed the manufacturer's instructions (Beyotime, China, C2006). Briefly, Morn2+ / - and Morn2- / - Mouse cauda epididymis, sperm was squeezed out from the cauda epididymis. Sperm was placed in 0.5 ml 1X JC-1 working solution prepared in advance and incubated at 37°C for 20 min. After washing three times by centrifugation with 1 ml JC-1 staining buffer (1X), it was suspended with 200 μl JC1 staining buffer (1X) and immediately imaged on a SP8 microscope (Leica). Fluorescence quantitative analysis was performed using ImageJ software. MMP analysis was repeated at least three times.

[0092] Sperm reactive oxygen species (ROS) level measurement

[0093] The protocol followed the manufacturer's instructions (Beyotime, China, S0033). Briefly, Morn2+ / - and Morn2- / -The cauda epididymis of mice was used to squeeze out sperm from the cauda epididymis. Spermatozoa were placed in a pre-made 0.5 ml DCFH-DA solution (10 μM / L) and incubated at 37°C for 20 min. After washing three times by centrifugation with PBS, they were suspended in PBS and immediately imaged on an SP8 microscope (Leica). Fluorescence quantitative analysis was performed using ImageJ software. ROS analysis was repeated at least three times.

[0094] Sperm ATP concentration measurement

[0095] The protocol followed the manufacturer's instructions (Beyotime, China, S0026). Briefly, Morn2+ / - and Morn2- / -Mouse cauda epididymis, squeeze out sperm from the cauda epididymis. Incubate the sperm in 200 μl of pre-prepared ATP lysis buffer, gently invert the lysis buffer several times to complete the reaction. After centrifugation, take the supernatant for subsequent measurement (sample lysis buffer is performed at 4°C or on ice). Dilute the ATP standard solution with ATP test lysis buffer into a graded series of 0.01, 0.03, 0.1, 0.3, 1, 3 and 10 μM. Add 100 μl of pre-prepared ATP test solution to each test well on a black 96-well plate. After 3-5 minutes of background ATP consumption, add 20 μl of sample or standard to the test well and mix quickly with a micropipette.

[0096] Each sample was replicated three times. After 3 min interval, the RLU value was measured by a photometer. The ATP concentration determination analysis was repeated at least three times.

[0097] Statistical analysis

[0098] All experiments were repeated at least 3 times, and the results are presented as mean ± SEM. The Student's t-test with paired two-tailed distribution was used to measure the statistical significance of the differences between the means of different genotypes. P < 0.05 was considered statistically significant.

[0099] Example 2 Morn2 is an evolutionarily conserved protein that is enriched in the testis

[0100] Through the Ensembl database (http: / / asia.ensembl.org / index.html), it was found that MORN-motif protein 2 is highly evolutionarily conserved in animals such as humans, chimpanzees, dogs, cows, rats, chickens, zebrafish, and frogs. Consistent with the expression profile in the Ensembl database (http: / / asia.ensembl.org / index.html), RT-PCR analysis of multiple organs of wild-type (WT) mice, including liver, spleen, lung, kidney, uterus, ovary, and testis, showed that Morn2 was specifically expressed in the testis. Developmental time series RT-PCR analysis of the testis was also performed, and it was found that the expression level of Morn2 in the testis was low starting from postnatal day 8 (PD8), which is roughly consistent with the generation of spermatogonia. Then the expression of Morn2 increased steadily, reaching a plateau and remaining stable from PD28, which roughly corresponds to the accumulation stage of haploid round sperm in spermatogenesis. Notably, this expression trend of Morn2 roughly coincided with the onset of spermatogenesis, suggesting that the testis-specific protein Morn2 may play a role in spermatogenesis.

[0101] Example 3 Morn2 gene knockout leads to male infertility.

[0102] In order to clarify the potential function of Morn2 in spermatogenesis, CRISPR / Cas9 technology was used to culture Morn2- / -mouse. The region from exon 4 to exon 5 was selected as the target site, and a 1841 bp deletion ( Figure 1 A). Genomic DNA extraction and PCR results of gene-edited mice confirmed the desired gene editing efficiency ( Figure 1 B) Heterozygous genome-edited mice have normal morphology and give rise to healthy offspring.

[0103] In Morn2- / - No obvious gross phenotype in mice: / Compared with Morn2- / - There were no significant differences between the mice in terms of testis size, mouse body weight, testis weight, and ratio of testis weight to mouse body weight ( Figure 1 CF). Male and female Morn2- / - Fertility of mice: Male individuals (Morn2+ / - and Morn2- / -) were housed with three wild-type (WT) females for 4 months and, at 9 a.m. on the second day of mating, were placed in Morn2+ / - and Morn2- / -Mating plugs were detected in both cages. Morn2- / - Male mice did not give birth ( Figure 1 G), while Morn2+ / -male mice successfully produced offspring, with an average litter size of 7.40±1.90 (n=3). Compared with this male infertility, it was found that when mating with WT males of similar age, Morn2- / - Female mice can produce offspring, but the average number of pups is low, (4.25 ± 2.25) per litter (n = 3), indicating that Morn2 may have an effect on female fertility but is not essential. Hematoxylin staining of ovarian sections showed no significant differences in ovarian morphology and follicular development.

[0104] Thus, MORN2 is essential for male fertility but not for female fertility.

[0105] Example 4Morn2- / - Sperm showing head inversion

[0106] Morn2- / -Hematoxylin staining of mouse testis and epididymis sections showed no obvious abnormalities in the morphology and development of seminiferous tubules; Morn2+ / - and Morn2- / - There was no difference in epididymal sperm count in mice ( Figure 2 AB). In Morn2+ / - and Morn2- / - In testis sections from mice, PAS staining detected all expected seminiferous tubule epithelial components and stages ( Figure 2 F) These results indicate that loss of Morn2 has no significant effect on early spermatogenesis.

[0107] Immunofluorescence staining was used to detect the acrosome biogenesis of PNA in sperm spread and found that Morn2+ / - and Morn2- / - There were no abnormalities between mice. Multiple immunofluorescence staining experiments were performed (using GM130 / GOPC as a marker for the Golgi apparatus) and Morn2+ / - and Morn2- / - mouse testes. These results suggest that the occurrence of acrosome / - In the testicles it is normal.

[0108] Subsequent hematoxylin and eosin staining showed that Morn2- / - Abnormal phenotype of mouse sperm: inverted head, curved flagellum and curled tail ( Figure 2 D) Morn2- / - The proportion of abnormal sperm was about 55.21±6.32(%), while Morn2+ / -The proportion of abnormal sperm in the group was only 11.28±1.41(%) ( Figure 2 D), Morn2- / - Group had a sperm ratio of 44.59±4.94(%), Morn2+ / - group, this data was only 3.75±1.78(%) (Figure E), and in Morn2+ / -Group and Morn2- / There was no significant difference in the proportion of sperm with curved flagella and curled tails in the Morn2- group of mice. These findings suggest that Morn2- / -Mice exhibit malformed sperm characterized by a curved head

[0109] TUNEL staining of testicular sections showed that Morn2+ / Compared with Morn2- / - Increased number of apoptotic cells in the seminiferous tubules of mice ( Figure 3A), these cells are concentrated in elongating sperm, suggesting that Morn2 may affect late spermatogenesis. Previous studies have reported that abnormalities in sperm morphology can interfere with sperm motility. In addition, abnormal curvature of sperm morphology is associated with mitochondrial sheath morphology disorders, although the mechanism of this abnormality is still unclear.

[0110] It can be seen that Morn2 plays a role in the late stage of spermatogenesis and affects the final morphology of sperm.

[0111] Example 5Morn2- / -Sperm motility is severely impaired

[0112] Next, we studied Morn2- / -Does abnormal sperm morphology affect motility? Computer-associated sperm analysis (CASA) shows that Morn2+ / -Compared to Morn2- / - The average path velocity (VAP), forward velocity (VSL) and trajectory velocity (VCL) of spermatozoa were significantly reduced ( Figure 3 BD). In addition, Morn2- / - mice, only 6.93% of sperm were motile and only 0.07% of sperm were rapidly motile. / -, these two values ​​were 71.97% and 7.97%, respectively (Figure EF). These results indicate that Morn2- / -Sperm motility is significantly reduced.

[0113] To study Morn2- / -Whether the sperm of mice undergo capacitation and acrosome reaction, and whether the male Morn2- / -mice and female WT were subjected to in vitro fertilization (IVF) analysis. It was observed that when intact oocytes were used for IVF, Morn2- / -Male mice can successfully complete fertilization ( Figure 3 G), despite the difference with Morn2+ / Compared with Morn2- / -mice have a reduced fertilization rate. This finding suggests that Morn2- / -mouse spermatozoa are able to penetrate the zona pellucida (ZP) and fuse with oocytes. / - and Morn2- / - male mice were mated with superovulated WT female mice for in vivo fertilization experiments, and the fertilization rate was determined by counting the number of two-cell embryos released from the ampulla of the oviduct 36 h after mating. / - No two-cell embryos were detected in females mated with males ( Figure 3 H), which indicates that Morn2- / -Sperm cannot complete fertilization in vivo. Therefore, although some Morn2- / - Spermatozoa can penetrate the zona pellucida (ZP) and fuse with oocytes, but they cannot complete fertilization in vivo, suggesting that Morn2- / - Infertility in mice may be caused by the inability of sperm to swim to the vicinity of the oocyte at the appropriate time rather than dysfunction of sperm-oocyte fusion, and the detected sperm motility defects can reasonably explain this abnormality.

[0114] The results showed that Morn2- / - Mouse sperm can penetrate the ZP and fuse with oocytes in vitro, but cannot complete fertilization in vivo due to their low motility, which ultimately causes male infertility.

[0115] Example 6Morn2- / - Abnormal mitochondrial morphology of mouse sperm

[0116] Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were used to characterize Morn2- / -defects in sperm were analyzed in detail by ultrastructure. Electron microscopy revealed that Morn2- / - The mitochondria of mouse sperm are hypertrophic and swollen, and the dense fibers in the outer layer of the middle section of the flagellum are disordered and proliferated; no obvious abnormalities were observed in the main section and the tail section ( Figure 4 A). Scanning electron microscopy analysis showed that Morn2+ / -Sperm compared to Morn2- / - Spermatozoa have different phenotypes, including inverted head, curled flagella, hypertrophy of mitochondrial sheath, and fragmented tail ( Figure 4 B).

[0117] Using high-power transmission electron microscopy to examine the longitudinal section of the middle of the flagellum: Morn2+ / -sperm mitochondria are tightly packed around the axoneme with regular arrangement and morphology, while Morn2- / - Mitochondria are irregularly arranged and fail to form a tight sheath around the axon ( Figure 4 C) Morn2- / - The mitochondria of mouse sperm are uneven in size and irregular in shape ( Figure 4 C) Analysis of the cross-section of the flagellar midsection showed that Morn2+ / - The mouse mitochondria fused into the expected tightly packed sheath-like structure, with the outer dense fibers neatly arranged within the mitochondrial sheath ( Figure 4 D) In ​​contrast, Morn2- / -The mitochondria of mice are arranged irregularly and loosely, with the outer dense fibers scattered radially within the mitochondrial sheath (Figure D).

[0118] These Morn2- / -Abnormal morphology and structure of sperm mitochondrial sheath is one of the causes of sperm motility defects and male infertility.

[0119] Example 7Morn2- / -Abnormal sperm energy metabolism

[0120] The decrease of mitochondrial membrane potential is considered to be a sign of early apoptosis and a manifestation of mitochondrial dysfunction. / -Whether sperm mitochondrial morphological abnormalities affect mitochondrial membrane potential was investigated using fluorescent cationic carbocyanine dye (JC-1) as a probe to investigate Morn2+ / - and Morn2- / -sperm were tested; this marks mitochondria with high membrane potential in red and mitochondria with low membrane potential in green. Morn2- / - The green signal of spermatozoa increases and the red / green signal ratio decreases, indicating that their membrane potential is low and mitochondrial function is impaired ( Figure 5 AB).

[0121] ROS can be produced by OXPHOS and is an important secondary messenger that regulates intracellular pathways through oxidative activation. Excessive ROS production can induce oxidative stress, thereby mediating sperm damage and male infertility. / - and Morn2- / - Sperm were analyzed for ROS levels ( Figure 5 C) found that Morn2- / - Upregulation of ROS levels in sperm ( Figure 5 D) These findings suggest that Morn2- / -The balance between ROS and antioxidants in mice is disrupted, causing oxidative stress.

[0122] Mitochondria produce ATP through OXPHOS to power sperm differentiation and motility. / - and Morn2- / -ATP was separated from sperm lysate and the concentration of ATP in the lysate was measured by photometer. / -The ATP concentration in sperm was significantly reduced by an order of magnitude, indicating that Morn2- / - Sperm aerobic respiration is severely impaired ( Figure 5E). In summary, knockout of Morn2 resulted in decreased sperm mitochondrial membrane potential, increased ROS levels, and decreased ATP concentration, indicating that Morn2 is required for normal sperm energy metabolism.

[0123] The inventors of this application have demonstrated for the first time that Morn2 is not only involved in the formation of the mitochondrial sheath, but also in the energy metabolism of sperm mitochondria. The research of this application reveals the potential pathogenic genes of asthenozoospermia and suggests potential targets for the development of male contraceptive technology. The inventors of this application have found that Morn2 regulates the morphology and energy metabolism of mitochondria and is necessary for male fertility. Morn2- / -Male mouse infertility, Morn2- / -Spermatozoa showed an abnormal phenotype of inverted head and extremely reduced motility. Abnormal formation of mitochondrial sheath detected by transmission electron microscopy and scanning electron microscopy, as well as abnormal energy metabolism characterized by low MMP, high ROS and significantly reduced ATP concentration, may be the cause of the above phenotypes. MORN2 was originally considered to be a protein required for the dynamic regulation of acrosome formation in the late spermatogenesis. In this study, in Morn2- / - mice, indicating that MORN2 is not required for acrosome formation. Morn2 is an evolutionarily conserved testis-enriched protein with two MORN motif domains. Previous studies have reported that junctophilin type 2 (JP2) can directly bind to the small conductance Ca2+ in mouse heart tissue through its N-terminal MORN motif. 2+ Activated K + SK2 interacts with SK2 channel subtype 2 (SK2) and regulates cardiac motility and Ca by modulating SK2 channel current amplitude and transport. 2+ Homeostasis. The MORN2 domain of JP2 was also found to enhance the expression of SK2 channels on the cell membrane and the density of Ik and ICa. Since SK2 is expressed at low levels in the testis, MORN2 has a potential interaction with SK2 in the testis, affecting electron transport in germ cells, thereby affecting sperm membrane potential and ultimately regulating sperm motility.

[0124] Applicants have thus provided methods for diagnosing fertility defects such as infertility disorders in mammals, including humans, and further facilitated the development of new treatments for male infertile individuals affected by gene mutations.

[0125] The above is an explanation of the present invention and cannot be regarded as a limitation of the present invention. Unless otherwise noted, the practice of the present invention will use the conventional techniques of organic chemistry, polymer chemistry, biotechnology, etc., and it is obvious that the present invention can also be realized in other ways except that it is particularly described in the above description and embodiments. Other aspects and improvements within the scope of the present invention will be apparent to those skilled in the art to which the present invention belongs. According to the teachings of the present invention, many changes and variations are feasible, and therefore they are within the scope of the present invention.

Claims

1. Use of a reagent for detecting MORN2 in a test individual for preparing a kit for diagnosing fertility defects such as infertility in mammals of different sexes.

2. The use according to claim 1, wherein the kit further comprises a reagent for detecting the gender of the subject.

3. The use according to claim 1, wherein the infertility disorder is selected from asthenozoospermia or asthenozoospermia.

4. The use according to any one of claims 1 to 3, wherein the detection reagent is a reagent for detecting the Morn2 gene.

5. The use according to any one of claims 1-3, comprising a reagent for detecting MORN2 protein, preferably, further comprising a reagent for determining the activity of MORN2 protein.

6. The use according to claim 5, which comprises a reagent for detecting the expression of MORN2 protein by immunoassay, such as a reagent for detecting the expression by ELISA or Western blotting with an antibody that specifically recognizes the protein; or These include reagents for detecting the presence or amount of mRNA of MORN2 protein, such as reagents for detecting the amount of mRNA encoding the protein in a sample by RT-PCR.

7. A kit for diagnosing fertility defects such as infertility in mammals of different sexes, comprising a reagent for detecting MORN2 in a subject and a reagent for detecting the sex of the subject.

8. The kit of claim 7, wherein the infertility disorder is selected from asthenozoospermia or asthenozoospermia.

9. The kit according to claim 7, wherein the reagent for detecting MORN2 in the subject is a reagent for detecting the Morn2 gene.

10. The kit according to claim 7, wherein the reagent for detecting MORN2 in the subject is a reagent for detecting MORN2 protein, and preferably, further comprises a reagent for measuring the activity of MORN2 protein.

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