Methods for detecting sperm

Abstract

To provide a novel method of more inexpensively staining sperm heads with higher sensitivity compared with conventional methods.SOLUTION: Sperm heads are visualized by staining sperm heads using a compound represented by a formula below. (R1, R2 represent the same or different 1 to 4 substituents on a benzene ring, each independently representing a hydrogen atom or a lower alkyl group, and R3, R4 represent a hydrogen atom or a lower alkyl group.)SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a method for detecting the presence or absence of sperm, morphological abnormalities, etc. in a sample.

Background Art

[0002] It is estimated that male infertility patients account for about 1% of all adult men. The criteria for normal sperm in the "Semen Examination Standardization Guidelines" formulated by the Japanese Urological Association dealing with male infertility in 2003 are "sperm with an oval head swimming", and the basic examination of semen (semen volume, sperm count, concentration, motility, confirmation of head and tail morphology) is the most important index in determining the treatment policy. Therefore, in a normal examination, a patient's semen specimen (semen smear, purified sperm smear, etc.) is observed under a microscope to confirm these elements. However, especially for the observation of head morphology, the diagnostic accuracy in ordinary outpatient clinics cannot be said to be high. The main reasons include the lack of an appropriate staining method for detecting sperm. For the microscopic observation of semen specimens (semen smear, purified sperm smear, etc.) in outpatient examinations, simple cell staining called Giemsa or Diff-Quik is usually performed. In specimens rich in normal sperm, sperm can be easily detected by these stainings. On the other hand, in specimens from infertility patients, there may be almost no normally shaped sperm, and furthermore, due to the large amount of cells other than sperm mixed in by semen concentration operations, their detection is extremely difficult.

[0003] Sperm contain a sperm-specific nuclear protein called "protamine". Since protamine is incorporated into the sperm nucleus at the end of the spermatogenesis process in the testis, the presence or absence of sperm and its maturity can be roughly determined by detecting protamine. In order to detect sperm from a specimen in which a large amount of cells other than sperm are mixed, immunostaining using an anti-protamine antibody is considered to be theoretically effective. However, immunostaining requires expensive antibodies and a working process of at least 3 hours, and it is not realistic to perform it in general outpatient examinations or rapid diagnosis during surgery. Furthermore, in the case of fluorescence detection, an expensive fluorescence microscope is required, and it is difficult to install in many laboratories.

[0004] As an alternative to immunohistochemistry using antibodies, a method has been disclosed in which sperm heads are stained and visualized using Reactive Blue 2, a staining dye containing an anthraquinone ring (Non-Patent Literature 1). The method using RB2 as the staining dye for sperm heads is considered to be more practical than immunohistochemistry because it does not require the use of anti-protamine antibodies and the procedure is very simple.

[0005] As described above, in addition to immunohistochemistry, reactive blue 2 staining has also been reported as a staining method for detecting sperm from a sample. However, there is a high need for a non-immunohistochemical staining method that can stain sperm heads with higher sensitivity compared to reactive blue 2 staining, and is also less expensive to increase versatility. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Kaneko et al., J. Med. Diagn. Meth. 2013, 2:6 Doi; 10.4172-2168-9784. 1000145 [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] In view of the above circumstances, the present invention aims to provide a new method for staining sperm heads with higher sensitivity and lower cost compared to conventional methods. [Means for solving the problem]

[0008] The inventors have discovered for the first time that Reactive Black 5, a type of reactive dye conventionally used as a dye for textiles and leather products, has a high affinity for protamine, a sperm-specific protein, in alkaline solutions, and that cell staining can sensitively and specifically detect the heads of protamine-containing spermatids in semen samples. The staining process for spermatids is simple, taking only about 10 minutes. Furthermore, we discovered that reactive Black 5 staining allows for detailed observation of sperm head morphology, particularly the presence and shape of intranuclear vacuoles, which were previously difficult to identify in conventional laboratory settings. We also confirmed that reactive Black 5 exhibits similar reactivity to sperm protamine in non-human mammals, such as mice and pigs.

[0009] In other words, the present invention is as follows (1) to (9). (1) A method for visualizing sperm heads by staining them with a compound represented by the following formula (I). [ka] (In formula (I), R1 represents one to four identical or different substituents on the benzene ring, each independently representing a hydrogen atom or a lower alkyl group; R2 represents one to four identical or different substituents on the benzene ring, each independently representing a hydrogen atom or a lower alkyl group; R3 represents a hydrogen atom or a lower alkyl group; and R4 represents a hydrogen atom or a lower alkyl group.) (2) The method according to (1) above, wherein R1, R2, R3 and R4 are hydrogen. (3) A method for detecting sperm, comprising visualizing the sperm head by the method described in (1) or (2) above. (4) A method for evaluating morphological abnormalities of sperm heads, comprising visualizing the sperm heads by the method described in (1) or (2) above. (5) The method according to (4) above, characterized in that the morphological abnormality is an abnormal vacuole. (6) A method for detecting protamine in a sample, comprising binding a compound represented by formula (I) above to protamine and visualizing the protamine. (7) The method according to (6) above, wherein R1, R2, R3, and R4 are hydrogen. (8) A reagent for visualizing sperm heads or protamine, comprising at least a compound represented by formula (I) above. (9) The reagent according to (8) above, wherein R1, R2, R3, and R4 are hydrogen. In this specification, the symbol "~" indicates a numerical range that includes the values ​​to its left and right. [Effects of the Invention]

[0010] The present invention provides a highly sensitive and inexpensive method for staining and visualizing sperm heads. Compared to conventional methods, the sperm head staining and visualization method according to the present invention allows for simpler and more sensitive detection of sperm heads. Therefore, by using the method of the present invention, it becomes possible to accurately determine the choice of treatment method for infertility, such as whether to apply or avoid highly invasive microdissection testicular sperm extraction (MD-TESE). [Brief explanation of the drawing]

[0011] [Figure 1] This shows the results of staining human sperm samples with Reactive Black 5 after a diagnosis of azoospermia. The stained sperm specimens were observed under a light microscope. The image on the left is at a magnification of 1,000x, and the multiple images on the right are representative examples of stained cells, also magnified at 1,000x. [Figure 2] This shows the results of staining a human sperm sample with Reactive Black 5 after being diagnosed with severe oligozoospermia. The stained sperm specimen was observed under a light microscope at a magnification of 1,000x. [Figure 3] This shows the results of staining sperm with Reactive Black 5 at different concentrations and comparing the contrast of the stained images. [Figure 4]The results of comparing the staining images of sperm with reactive black 5 and reactive blue 2 are shown. Left figure: staining image with 0.001% reactive black 5, right figure: staining image with 0.02% reactive blue 2. [Figure 5] The results of binarizing the staining image of the human sperm head with reactive black 5 are shown. The sperm smear staining images stained with reactive blue 2 (upper row) and reactive black 5 (lower row) were converted into grayscale images using ImageJ, then binarization processing was performed, and the number of areas recognized as "cells" on the image was counted by automatic cell recognition. [Figure 6] Examination of the detection of human sperm head vacuoles by reactive black 5 staining. Since the head vacuoles that became white with reactive black 5 (left panel, untreated) disappeared after 1 mM DTT treatment (right panel, 1 mM DTT treatment), it was inferred that the structures around the vacuoles were those whose structures were broken by the reducing agent treatment, that is, protamine, and the vacuole part was a region where protamine condensation was impaired. [Figure 7] Transmission electron microscopy image of sperm with head vacuoles (upper left panel, TEM). The vacuole part is filled with chromatin-like structures with low electron density. Immunostaining images of sperm with head vacuoles (upper right, lower left and lower panels). The vacuole part (arrow) is sparse in DNA (upper right) and protamine 2 (PRM2) (lower right) compared with the surrounding area, while histone H3 is condensed (lower left). [Figure 8] The results of observing the morphology of the human sperm head by reactive black 5 staining are shown. Sperm specimens (raw semen and purified sperm) with mild to severe morphological abnormalities and nuclear vacuole rates were stained with reactive black 5. [Figure 9] The results of detecting human protamine with reactive black 5 are shown. Protamine was extracted from human sperm, acrylamide electrophoresis was performed, and then staining was performed with reactive black 5 (right figure). As a control, CBB (Coomassie Brilliant Blue) staining was performed (left figure). [Figure 10] The staining results of mouse testicular sections with reactive black 5 are shown.

Mode for Carrying Out the Invention

[0012] Hereinafter, embodiments for carrying out the present invention will be described. The first embodiment is a method of visualizing sperm heads by staining sperm heads with a compound represented by the following formula (I) (hereinafter also referred to as "the visualization method according to the present embodiment").

Chemical formula

[0013] Reactive Black 5, a representative compound of formula (I), specifically binds to protamine present in sperm heads under alkaline conditions. When Reactive Black 5 binds to protamine in sperm heads, it stains the sperm heads dark blue. Therefore, when Reactive Black 5 is added to a sample containing sperm, the sperm heads become stained dark blue and visible. The visualized sperm heads can then be clearly observed under a microscope. Furthermore, the staining state of sperm heads can be captured using a camera or similar device, images of the stained sperm heads can be obtained, and these images can be digitized to quantify their staining intensity. For example, the color of the stained sperm heads can be separated into red, green, and blue RGB colors using a bioimaging analysis system, and the staining properties of each color can be binarized according to their hue.

[0014] The second embodiment is a method for detecting sperm, which includes visualizing the sperm head by staining it with a compound represented by formula (I) (hereinafter also referred to as "the detection method according to this embodiment"). That is, it is a method for detecting sperm by visualizing the sperm head using the visualization method according to the first embodiment. The samples to be tested in this embodiment include, but are not limited to, semen and semen specimens obtained from animals (humans and non-human animals) (e.g., semen smears, purified sperm smears, testicular tissue sections, etc.). The detection method in this embodiment includes a step of contacting a sample such as a semen specimen with a dye compound of formula (I). This step is not particularly limited, but can be carried out by adding a solution containing the dye compound of formula (I) (e.g., an alkaline solution (e.g., pH 9 to pH 11, preferably pH 10)) to the semen specimen and incubating (e.g., letting it stand) at room temperature for several minutes to an hour. The conditions listed here (alkaline conditions, temperature, standing time, etc.) are merely examples, and appropriate conditions can be selected by those skilled in the art through appropriate preliminary experiments. After contacting the semen sample with the dye compound of formula (I) and incubating, any excess dye compound of formula (I) is removed by washing or other means, and then the presence or absence of stained sperm heads is observed using a microscope or other means. After treating the sample with the dye compound of formula (I), if sperm are present in the sample, their heads will be stained and visualized, allowing for detection.

[0015] In the detection method according to this embodiment, the concentration of the dye compound of formula (I) that is brought into contact with the sample can be appropriately selected by a person skilled in the art through preliminary experiments, but to give an example, if the dye compound of formula (I) is an aqueous solution of reactive black 5, the concentration is 0.0001% (w / v) to 0.001% (w / v).

[0016] Compared to conventional immunohistochemical staining methods, the detection method according to this embodiment allows for the rapid, simple, and highly sensitive detection of sperm heads. Therefore, by using the detection method according to this embodiment, it is possible to quickly and accurately determine the presence or absence of sperm in a sample from an infertile patient, and furthermore, to roughly determine the maturity of the sperm. As a result, it becomes possible to accurately decide on the choice of treatment method, such as whether to apply or avoid highly invasive microdissection testicular sperm extraction (MD-TESE).

[0017] The third embodiment is a method for evaluating morphological abnormalities of sperm heads, which includes visualizing the sperm heads by staining them with a compound represented by formula (I) above (hereinafter also referred to as "the evaluation method according to this embodiment"). That is, it is a method for visualizing sperm heads and evaluating morphological abnormalities of sperm heads according to the first embodiment. Even in normal sperm heads, small vacuoles exist at physiological levels within the sperm head (sperm nucleus). When these vacuoles are numerous or enlarged, occupying more than one-third of the nuclear volume, they are called abnormal vacuoles (abnormal sperm). The inside of the vacuole is observed as a region of low electron density under a transmission electron microscope, suggesting localized chromatin condensation failure. Reactive Black 5, one of the compounds in formula (I), binds to protamine, so vacuoles lacking protamine are observed as white holes. Therefore, abnormal vacuoles can be identified even under a normal microscope by staining with Reactive Black 5.

[0018] While abnormal vacuoles are considered clear morphological abnormalities, there is currently no consensus on whether these morphological abnormalities have a functionally adverse effect on pregnancy. However, some reports suggest that intraspermal vacuoles have adverse effects at every stage from fertilization to the next generation, including decreased early embryonic development, decreased implantation rates, increased miscarriage rates, increased premature birth rates, and even an increased birth rate of children with congenital defects (Cassuto et al., Fertil Steril. 2009 Nov;92(5):1616-25. doi: 10.1016 / j.fertnstert.2008.08.088.; Boitrelle et al., Hum Reprod. 2011 Jul;26(7):1650-8. doi: 10.1093 / humrep / der129.). Therefore, by evaluating the presence or absence of abnormal sperm head morphology using the evaluation method according to this embodiment, it may be possible to predict the possibility of adverse effects after fertilization, such as a decrease in early embryonic development. Furthermore, sperm head inversion and intranuclear vacuoles, which are physiological changes often observed in sperm, are difficult to distinguish under low-resolution microscopy. In contrast, the evaluation method according to this embodiment makes it possible to distinguish between sperm head inversion and abnormal vacuoles, which are physiological changes.

[0019] The fourth embodiment is a method for detecting protamine in a sample, which includes binding a compound represented by formula (I) to protamine and visualizing the protamine. Protamine is a sperm-specific nuclear protein thought to be involved in sperm nucleus condensation and decondensation after entry into the egg. There are four types of protamine (P1, P2, P3, P4), of which P1 and P2 are the main components of protamine found in sperm. In recent years, many abnormalities in the sperm P1 / P2 ratio have been reported in cases of male infertility (e.g., Aoki et al., Fertil Steril. 2006 Nov;86(5):1408-15. doi: 10.1016 / j.fertnstert.2006.04.024. Epub 2006 Sep 29.; Amor et al., International Journal of Women's Health and Reproduction Sciences Vol. 6, No. 4, October 2018, 400-409). Most of these reports are based on results obtained by Western blotting, which separately detects P1 and P2 with their respective specific antibodies. Conventional Western blotting methods for detecting P1 and P2 require cumbersome procedures and are time-consuming and costly to obtain results.

[0020] Reactive Black 5 is useful for detecting protamine because it specifically binds to it. For example, after performing gel electrophoresis on a protamine-containing sample (e.g., a sample extracted from sperm heads), Reactive Black 5 can be bound to protamine simply by immersing the gel directly in a Reactive Black 5 solution. As a result, P1 and P2 are detected on the gel as dark blue bands. By quantifying the staining intensity of the detected bands using densitometry or other methods, the relative abundance of P1 and P2 can be quantified. Compared to conventional detection methods using protamine antibodies, this method significantly reduces time, effort, and cost. Furthermore, since P1 and P2 are separated into two bands on the same lane, there is no need to detect P1 and P2 separately as in Western blotting. This avoids technical artifacts that can greatly affect the results, such as sample application errors and transfer inconsistencies, and allows for more accurate measurement of the P1 / P2 ratio.

[0021] The fifth embodiment is a reagent for visualizing sperm heads or protamine, comprising at least a compound represented by formula (I) above. The reagent according to this embodiment may include the compound of formula (I), such as Reactive Black 5, as well as additives for reagent preparation. The reagent according to this embodiment is not particularly limited, but may be in the form of a liquid, powder, or solid (tablet, pill, etc.), and may contain components such as pH adjusters and preservatives as needed. To produce solid reagents, for example, the active ingredient, compound (I), may be mixed with excipient components to form a powder, or binders, disintegrants, etc., may be added and wet or dry granulated to produce granules. To produce tablets, these powders and granules may be used as is, or a lubricant may be added and compressed into tablets.

[0022] In embodiments of the present invention, "sperm" and "protamine" include all those derived from humans and non-human animals. Here, non-human animals include, but are not limited to, fish such as salmon and herring, pet animals such as dogs, cats, and rabbits, and domestic animals such as cows, pigs, sheep, and horses.

[0023] Where this specification is translated into English and contains the singular forms of "a," "an," and "the," it shall be understood to include both singular and plural forms unless the context clearly indicates otherwise. The present invention will be further explained below with reference to examples, but these examples are merely illustrative of embodiments of the present invention and do not limit the scope of the present invention. [Examples]

[0024] 1. Experimental Method 1-1. Staining of sperm heads with Reactive Black 5 Sperm smears (semen, washed sperm, fractionated sperm, etc.) should be obtained by visiting the Department of Obstetrics and Gynecology at Ichikawa General Hospital. The samples were obtained from male patients who consented to their use. Reactive Black 5 was dissolved in milli Q (Cat# 306452, Dye content ≥ 50%) purchased from Sigma-Aldrich to prepare a 0.1% (w / v) preservation solution. Before use, the preservation solution was diluted 100-fold with 0.1 M carbonate-bicarbonate buffer (pH 10). The specimen on the slide glass was outlined with a pap pen, and an appropriate amount of 0.001% (w / v) Reactive Black 5 was mounted in that area (the specimen area). The slide was left to stand at room temperature for 10 minutes. After that, the excess dye on the specimen was washed off with water, the slide was mounted on a coverslip, and then observed under a microscope.

[0025] 1-2. Binarization of stained images The TIFF image, captured under a microscope, was opened in ImageJ and converted to an 8-bit grayscale image. The upper and lower threshold values ​​of this image were then adjusted to set values ​​that would allow individual sperm to be distinguished from the background without being whited out or blacked out, and the image was then binarized.

[0026] 1-3. Staining of mouse testicular sections with Reactive Black 5 Mouse testes were fixed in formalin, dehydrated with an alcohol series, and embedded in paraffin. Thin sections were prepared using a microtome, deparaffinized, and then mounted with an appropriate amount of 0.01% (w / v) Reactive Black 5. The sections were left to stand at room temperature for 60 minutes. After that, excess dye on the specimens was washed off with water, mounted on coverslips, and then observed under a microscope.

[0027] 1-4. Extraction of sperm protamine After mixing the sperm suspension and extract in a 1:2 ratio, 1 / 100 volume of 500 mM DTT was added and mixed. Since it immediately gelled, it was left to stand for about 5 minutes. The prepared solutions used here were made as follows: extract liquid 0.5M Na2CO3- NaHCO3 pH 10.0 10 M urea 0.225 M Na2SO4 The extract with the above composition was prepared by diluting 40 mL of 0.5 M Na2CO3-NaHCO3 pH 10.0 (10.6 g of Na2CO3 and 8.4 g of NaHCO3 dissolved in 400 mL of water), 60 g of urea, and 3.2 g of Na2SO4 to 100 mL with water. 500 mM DTT 500 mM DTT 20mM acetate buffer pH 4.7 The above DTT stock was prepared using 1.0 M acetate buffer (prepared by mixing equal parts of 60 g (1.049 g / ml, 57 ml) / L) of acetic acid and 82 g / L of sodium acetate).

[0028] To three volumes of the gelled extract, one volume of 6.0% benzalkonium chloride 40% ethanol solution (final concentration 1.5%) was added to promote the dissociation of the DNA / protamine complex, and the mixture was stirred with a vortex mixer. Upon stirring, the DNA precipitated in thread-like structures and condensed. The condensed DNA was collected and removed by centrifugation or by picking it up with tweezers. Subsequently, to precipitate protamine, an equal volume of ethanol was added to the obtained extract, and the mixture was stirred with a vortex mixer. After centrifugation, the protamine was collected as a white precipitate. The supernatant after centrifugation was removed by decantation, and the small amount of 50% ethanol accumulated at the bottom was removed with a pipette.

[0029] 1-5. Electrophoresis of sperm protamine To prevent the reassociation of the SH groups of extracted protamine, electrophoresis was performed under acidic conditions. The pI of protamine is 11-12. Protamine is composed of almost a single amino acid, arginine, and is a basic protein; therefore, its charge does not change with pH, ​​and its mobility is not affected by pH. For electrophoresis, the concentrating gel was omitted, and only an 18% acrylamide (24:1) separation gel was prepared. The sample buffer was added to the protamine precipitate. The composition of the sample buffer is as follows: Sample buffer 7.0 M Urea 0.1 M Na2CO3- NaHCO3, pH 10.0 Before use, add 1 / 100 volume of 500mM DTT. After adding the sample buffer to the protamine precipitate, the mixture was stirred with a vortex mixer to dissolve the protamine. Any insoluble matter was removed by centrifugation.

[0030] The composition of the acrylamide gel used for separation and the buffer solution used for electrophoresis is shown below. Gel preparation mixture 77.76 g of acrylamide and 3.24 g of N,N'-methylenebis(acrylamide) were dissolved in 300 mL of water. Separation gel buffer To 114 mL of glacial acetic acid, NaOH solid was added to adjust the pH to 3.8-4.0, and then the solution was diluted to 1000 mL with water to prepare a 2.0 M acetate buffer solution. 18% Acrylamide Gel (45 mL) Gel preparation mixture (25 mL), separation gel buffer (2.0 M acetate buffer (pH 3.8)) (11.2 mL, final concentration 0.5 M), 10% APS (Ammonium Peroxodisulfate) (580 μL), and TEMED (N,N,N',N'-Tetramethylethylene-Diamine) (90 μL) were mixed with water to make up 45 mL, and after mixing, the mixture was allowed to stand at 36°C for at least 30 minutes. electrophoresis buffer (1) Lower electrophoresis buffer 0.3 M acetate buffer pH 4.8 was used. (2) Upper electrophoresis buffer Glycine (30g) and glacial acetic acid (0.3ml) were mixed with water to make a total volume of 2 L.

[0031] In this example, a gel with a thickness of 1.5 mm was used. The electrophoresis tank should be cooled using an ice bath. Electrophoresis conditions First stage (concentration): Constant current 60mA Second stage (separation) constant current 60mA Electrophoresis was performed for approximately 3.0 hours under the above conditions. The electrophoresis conditions can be set by those skilled in the art through preliminary experiments. For good separation of protamines P1 and P2, electrophoresis may be performed as follows, for example: After confirming that the electrophoresis front marker (CV: crystal violet), which has been sufficiently concentrated, has entered the gel to a depth of approximately 5 mm (first stage (concentration)) by electrophoresis at 60 mA (CC) for about 30-40 minutes, electrophoresis is stopped. One-tenth of the upper electrophoresis buffer is removed, and 20 ml of 1.0 M sodium acetate, equivalent to the amount removed, is added to make the upper electrophoresis buffer a 0.10 M sodium acetate solution. Then, electrophoresis is performed at 60 mA (CC) (second stage (separation)).

[0032] 1-6. Staining of protamine with Reactive Black 5 Preparation of staining solution 0.2% Reactive Black 5 and 0.1 M Na2CO3-NaHCO3 pH 10.0 were prepared as a stock staining solution. Before use, NaOH was added dropwise to this stock solution to adjust the pH to 10.7. After pH adjustment, the acrylamide gel on which protamine had been electrophoresed was immersed in the staining solution, left to stand at room temperature for 30-40 minutes, then immersed in water and left to stand overnight to decolorize. Because Reactive Black 5 is highly hydrophilic, decolorization is sufficient by simply immersing in water, but for faster decolorization, for example, immersion in 10% isopropanol / water may be used. For comparison, CBB staining was performed in 0.2% CBB (50% methanol / water). Specifically, the acrylamide gel was sandwiched between mesh sheets and immersed in the above CBB staining solution, and left to stand for 30-40 minutes. After that, the acrylamide gel sandwiched between mesh sheets was immersed in water, an appropriate amount of weakly basic anion exchange resin was added, and the mixture was gently stirred with a stirrer and destained overnight.

[0033] 2.Results 2-1. Confirmation of Reactive Black 5 staining properties using semen samples. Traditionally, when cells with tails (sperm) were not observed under a phase-contrast microscope, azoospermia was diagnosed, and then semen was centrifuged and the concentrated sediment was observed. Furthermore, methods using tissue staining such as Papanicolaou and Giemsa stains have been attempted. However, under a phase-contrast microscope, transparent particles overlap, making it difficult to observe sperm. Also, when staining with Papanicolaou or Giemsa stains, all cells (particles) are stained, making observation difficult in this case as well. Moreover, the most significant problem with conventional methods is that spermatocytes that have stopped forming tails during spermatogenesis, i.e., spermatocytes whose formation has stopped before clear tail formation, cannot be observed. This invention enables specific staining of protamine. When a concentrated sample is stained with Reactive Black 5, unstained particles are observed (Figure 1 left and Figure 2), while only cells containing protamine are stained blue (Figure 1 right and Figure 2), making it possible to clearly observe cells containing protamine. These results suggest that among cases previously diagnosed as azoospermia, there are cases in which the highly invasive MD-TESE procedure can be avoided.

[0034] On the other hand, among the cases examined, while sperm (or sperm-like cells) that stained with Reactive Black 5 were present, their morphology was so severely impaired that they were not immediately recognizable as sperm (Figure 1, right). In other words, sperm-like cells containing protamine but not normally recognized as sperm under a microscope, or protamine-containing degenerated spermatids referred to as "residual bodies," were detected by Reactive Black 5 staining. While it is not appropriate to use the aforementioned abnormal sperm for intracytoplasmic sperm injection (ICSI), the presence of protamine indicates that spermatogenic dysfunction occurred at least after protamine incorporation. In other words, these cases suggest a high probability of the presence of relatively immature spermatids within the testis, making them promising candidates for MD-TESE. Conversely, in cases where there are no reactive Black 5-positive sperm in the semen smear, the likelihood of obtaining sperm through MD-TESE is lower. Furthermore, the results shown in Figures 1 and 2 suggest that reactive Black 5 staining is also effective in sperm detection during MD-TESE. In other words, during MD-TESE, the visualization method of sperm using reactive Black 5 staining can help in rapidly determining the presence or absence of sperm (or sperm-like cells) that can be used for intracytoplasmic sperm injection (ICSI).

[0035] When sperm were stained with different concentrations of Reactive Black 5, it was found that higher concentrations resulted in clearer contrast in the stained image, but the color removal of vacuoles (white areas) was poorer (Figure 3). Therefore, it is desirable to appropriately select the concentration of Reactive Black 5 to be used depending on the purpose of observation.

[0036] Next, we compared the stained images of sperm using Reactive Blue 2 (0.05%) and Reactive Black 5 (0.001%) (Figure 4). As a result, it was confirmed that staining with Reactive Black 5 resulted in clearer contrast in the stained image at low concentrations. Next, the stained images of sperm using Reactive Blue 2 (0.05%) and Reactive Black 5 were binarized, and their staining characteristics were compared (Figure 5). The upper and lower limits were adjusted so that almost all sperm visible in the stained image remained visible without blackout or whiteout, and then the images were binarized. These were counted using an automated cell recognition function, and the difference from the visually observed sperm count was compared. As a result, Reactive Blue 2 included more stained somatic cells in the count, resulting in a discrepancy of 9.17 times compared to the visually observed sperm count. On the other hand, Reactive Black 5 showed almost no staining effect on somatic cells, so the difference from the visually observed sperm count was only 2.64 times. The discrepancy with the actual number in Reactive Black 5 staining is because the staining intensity within a single sperm was counted as multiple sperm during automated measurement. We believe that this can be brought closer to the actual number by setting a definition (minimum to maximum area) for counting "one cell".

[0037] 2-2. Visualization of sperm head morphological abnormalities using Reactive Black 5 Even in normal sperm, small vacuoles at physiological levels exist in the sperm head. However, when these vacuoles are numerous or enlarged, occupying more than one-third of the sperm nucleus volume, they are called abnormal vacuoles (abnormal sperm). The inside of the vacuole is observed as a region with low electron density under a transmission electron microscope, suggesting localized chromatin condensation failure. Vacuoleous regions where protamine does not bind and chromatin condensation failure occurs are observed as white areas without staining with Reactive Black 5. In Figure 6, sperm heads treated with 1 mM DTT, which loosens the crosslinks between protamine molecules, show the disappearance of vacuoles and a decrease in staining with Reactive Black 5 (Figure 6, "1 mM DTT treatment"). When sperm heads were stained with Reactive Black 5 without DTT treatment, white areas were observed (Figure 6, "No Treatment"). These white areas are thought to be vacuolated due to the absence of protamine and resulting chromatin condensation failure. This hypothesis is indeed supported by the fact that transmission electron microscopy images of head vacuolated sperm show that the vacuoles are filled with chromatin-like structures with low electron density rather than condensed protamine (TEM in Figure 7), and further by the fact that immunohistochemical staining images of head vacuolated sperm show that the vacuolar region (arrow) has less DNA (upper right in Figure 7) and protamine 2 (PRM2) (lower right in Figure 7) compared to the surrounding area, while histone H3 is condensed (lower left in Figure 7). From the above, vacuoles present in the sperm head containing abnormal vacuoles appear white when stained with Reactive Black 5 and can be detected even with a regular microscope (Figure 6). Furthermore, Figure 8 shows that abnormalities in the shape of the sperm head can also be clearly detected by Reactive Black 5 staining.

[0038] 2-3. Examination of the relative abundance of protamine P1 and protamine P2 using reactive black 5 staining. Reactive Black 5 specifically binds to protamine. Therefore, it can be widely used as a substitute for conventional research and diagnostic applications that use anti-protamine antibodies. Protamine was extracted from human sperm and subjected to native acrylamide gel electrophoresis under acidic conditions. When the gel was stained with Reactive Black 5 after electrophoresis, protamine P1 and protamine P2 were detected as dark blue bands on the gel (Figure 9).

[0039] 2-4. Applicability of Reactive Black 5 staining to non-human semen analysis, etc. Since protamine is a protein found in the sperm of most animals, from fish to higher mammals, Reactive Black 5, as a substitute for anti-protamine antibodies, has broad applications in sperm research and industry for these animals. Currently, it has been confirmed that Reactive Black 5 actually binds to protamine in salmon and herring (fish), mice, and pigs. As an example, Figure 10 shows the results of visualizing mouse testicular sperm using Reactive Black 5 staining. [Industrial applicability]

[0040] This invention, as an alternative method for detecting protamine using an anti-protamine antibody, has broad applicability not only to medical applications for male infertility but also to applications in the livestock industry and basic research. Therefore, this invention is expected to be used in a wide range of fields, including livestock farming and medicine.

Claims

1. A method for visualizing sperm heads by staining them with a compound represented by the following formula (I). 【Chemistry 1】 (In formula (I), R １ R consists of one to four identical or different substituents on the benzene ring, each independently representing a hydrogen atom or a lower alkyl group. ２ R consists of one to four identical or different substituents on the benzene ring, each independently representing a hydrogen atom or a lower alkyl group. ３ R represents a hydrogen atom or a lower alkyl group. ４ (represents a hydrogen atom or a lower alkyl group.)

2. The aforementioned R １ , R ２ , R ３ and R ４ The method according to claim 1, wherein is hydrogen.

3. A method for detecting sperm, comprising visualizing the sperm head by the method according to claim 1 or 2.

4. A method for evaluating morphological abnormalities of sperm heads, comprising visualizing the sperm heads by the method described in claim 1 or 2.

5. The method according to claim 4, characterized in that the morphological abnormality is an abnormal vacuole.

6. A method for detecting protamine in a sample, comprising binding a compound represented by the following formula (I) to protamine and visualizing the protamine. 【Chemistry 2】 (In formula (I), R １ represents one to four identical or different substituents on the benzene ring, each independently representing a hydrogen atom or a lower alkyl group, and R ２ represents one to four identical or different substituents on the benzene ring, each independently representing a hydrogen atom or a lower alkyl group, and R ３ represents a hydrogen atom or a lower alkyl group, and R ４ represents a hydrogen atom or a lower alkyl group)

7. The aforementioned R １ , R ２ , R ３ and R ４ The method according to claim 6, wherein is hydrogen.

8. A reagent for visualizing sperm heads or protamine, comprising at least a compound represented by the following formula (I). 【Transformation 3】 (In formula (I), R １ R consists of one to four identical or different substituents on the benzene ring, each independently representing a hydrogen atom or a lower alkyl group. ２ R consists of one to four identical or different substituents on the benzene ring, each independently representing a hydrogen atom or a lower alkyl group. ３ R represents a hydrogen atom or a lower alkyl group. ４ (represents a hydrogen atom or a lower alkyl group.)

9. The aforementioned R １ , R ２ , R ３ and R ４ The reagent according to claim 8, wherein is hydrogen.

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