JP2023133892A - Sperm detection method - Google Patents

Abstract

The present invention provides a new method for staining sperm heads with higher sensitivity and at lower cost than conventional methods.
[Solution] The sperm head is visualized by staining the sperm head with a compound represented by the following formula.

(R ₁ and R ₂ are the same or different 1 to 4 substituents on the benzene ring, each independently representing a hydrogen atom or a lower alkyl group, and R ₃ and R ₄ are a hydrogen atom or a lower alkyl group. (represents a group)
[Selection diagram] Figure 1

Description

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

It is estimated that male infertility patients account for approximately 1% of all adult men. The criteria for normal sperm in the ``Semen Test Standardization Guidelines'' formulated in 2003 by the Japanese Urological Association, which deals with male infertility, is ``sperm with oval heads that are swimming.'' Semen volume, sperm count, concentration, motility, confirmation of head and tail morphology) are the most important indicators in determining treatment policy. Therefore, in normal examinations, a patient's semen specimen (semen smear, purified sperm smear, etc.) is observed under a microscope to confirm these factors, but the diagnostic accuracy in ordinary outpatient treatment is not particularly high when it comes to head morphology observation. I can not say. The main reasons include the lack of suitable staining methods that can detect sperm.
For microscopic observation of semen specimens (semen smear, purified sperm smear, etc.) in outpatient examinations, simple cell staining called Giemsa or DiffQuick is usually applied. In specimens with abundant normal sperm, spermatozoa are easily detected by these stains. On the other hand, samples from infertile patients may contain almost no normal spermatozoa, and furthermore, a large amount of non-sperm cells are mixed in during the semen concentration procedure, making detection extremely difficult.

Sperm contains a sperm-specific nuclear protein called "protamine." Protamine is incorporated into the sperm nucleus at the end of the spermatogenesis process in the testes, so by detecting protamine it is possible to roughly determine the presence or absence of sperm and their maturity. Immunostaining using an anti-protamine antibody is theoretically considered to be effective in detecting sperm from a specimen containing a large amount of cells other than sperm. However, immunostaining requires expensive antibodies and a process that takes at least three hours, making it impractical for general outpatient testing or rapid diagnosis during treatment. Furthermore, fluorescence detection requires an expensive fluorescence microscope, which is difficult to install in many examination rooms.

As a method other than immunostaining with antibodies, a method of staining and visualizing sperm heads with Reactive Blue 2, which is a staining dye having an anthraquinone ring, has been disclosed (Non-Patent Document 1). The method of using RB2 as a staining dye for sperm heads is considered to be more useful than immunostaining in that it does not use anti-protamine antibodies and the working process is very simple.

As described above, in addition to immunostaining, a method using reactive blue 2 staining has been reported as a staining method for detecting sperm from a specimen. However, there is a strong need for a non-immunostaining method that is capable of staining sperm heads with higher sensitivity than Reactive Blue 2 staining and is less expensive in order to increase versatility.

Kaneko et al., J. Med. Diagn. Meth. 2013, 2:6 Doi; 10.4172-2168-9784. 1000145

In view of the above circumstances, an object of the present invention is to provide a new method for staining sperm heads with higher sensitivity and at lower cost than conventional methods.

The inventors discovered that Reactive Black 5, a type of reactive pigment conventionally used as a dye for textiles and leather products, has a high affinity for the sperm-specific protein protamine in an alkaline solution. We have discovered for the first time that the heads of protamine-containing sperm cells contained in semen samples can be detected with high sensitivity and specificity by cell staining. The staining time in the sperm cell staining process is simple and only takes about 10 minutes.
Furthermore, we have found that Reactive Black 5 staining allows detailed observation of the morphology of sperm heads, particularly the presence or absence and shape of intranuclear vacuoles, which has been difficult to identify in conventional laboratories. It was also confirmed that Reactive Black 5 has similar reactivity to sperm protamine from mammals other than humans, such as mice and pigs.

That is, the present invention includes the following (1) to (9).
(1) A method of visualizing the sperm head by staining the sperm head with a compound represented by the following formula (I).
(In formula (I), R ₁ is the same or different 1 to 4 substituents on the benzene ring, each independently representing a hydrogen atom or a lower alkyl group, and R ₂ is the same or different on the benzene ring. 1 to 4 substituents, each independently representing a hydrogen atom or a lower alkyl group, R ₃ represents a hydrogen atom or a lower alkyl group, R ₄ represents a hydrogen atom or a lower alkyl group)
(2) The method according to (1) above, wherein R ₁ , R ₂ , R ₃ and R ₄ are hydrogen.
(3) A method for detecting sperm, which comprises visualizing the sperm head by the method described in (1) or (2) above.
(4) A method for evaluating morphological abnormalities in sperm heads, which comprises visualizing the sperm heads by the method described in (1) or (2) above.
(5) The method according to (4) above, wherein the morphological abnormality is an abnormal vacuole.
(6) A method for detecting protamine in a sample, the method comprising binding a compound represented by the above formula (I) to protamine and visualizing the protamine.
(7) The method according to (6) above, wherein R ₁ , R ₂ , R ₃ and R ₄ are hydrogen.
(8) A reagent for visualizing sperm heads or protamine, which contains at least a compound represented by the above formula (I).
(9) The reagent according to (8) above, wherein R ₁ , R ₂ , R ₃ and R ₄ are hydrogen.
Note that in this specification, the symbol "~" indicates a numerical range that includes the values on the left and right sides thereof.

The present invention provides a highly sensitive and inexpensive method for staining and visualizing sperm heads. The method for staining and visualizing sperm heads according to the present invention allows sperm heads to be detected easily and with high sensitivity compared to conventional methods. Therefore, by using the method of the present invention, infertility treatment strategies, such as whether to apply or avoid physically invasive microdissection testicular sperm extraction (MD-TESE), etc. It becomes possible to accurately judge the selection of treatment methods.

The results of staining a human sperm specimen diagnosed with azoospermia with Reactive Black 5 are shown. The sperm specimens after staining were observed under a light microscope. The figure on the left is at 1,000x magnification, and the multiple figures on the right are representative examples of stained cells at 1,000x magnification. The results of staining a human sperm specimen diagnosed with severe oligozoospermia with Reactive Black 5 are shown. The sperm specimens after staining were observed under a light microscope. The magnification is 1,000x. The results of staining spermatozoa with different concentrations of Reactive Black 5 and comparing the contrast of the stained images are shown. The results of comparing the stained images of sperm with Reactive Black 5 and Reactive Blue 2 are shown. Left image: Image stained with 0.001% Reactive Black 5, right image: Image stained with 0.02% Reactive Blue 2. The results of binarizing an image of a human sperm head stained with Reactive Black 5 are shown. Sperm smear images stained with Reactive Blue 2 (upper row) and Reactive Black 5 (lower row) were converted to grayscale images using ImageJ, binarized, and automatically recognized as "cells" on the images. ” was counted. Examination of detection of human sperm head vacuole by reactive black 5 staining. The head vacuole, which turned white with Reactive Black 5 (left panel, no treatment), disappeared with 1 mM DTT treatment (right panel, 1 mM DTT treatment), so the structures around the vacuole were treated with a reducing agent. It was inferred that the structure of protamine is destroyed by the process, and that the vacuole region is a region where protamine condensation is impaired. Transmission electron microscopy image of head-vacuolated sperm (upper left panel, TEM). The vacuole is filled with a chromatin-like structure with low electron density. Immunostaining images of head-vacuolated spermatozoa (upper right, lower left and lower panels). In the vacuole region (arrow), DNA (top right) and protamine 2 (PRM2) (bottom right) are sparse compared to the surrounding area, while histone H3 is condensed (bottom left). The results of observing the morphology of human sperm heads by Reactive Black 5 staining are shown. Sperm specimens (original semen and purified sperm) in which morphological abnormalities and intranuclear vacuole rates were determined to be mild to severe were stained with Reactive Black 5. The results of detection using Reactive Black 5 of human protamine are shown. Protamine was extracted from human sperm, subjected to acrylamide electrophoresis, and then stained with Reactive Black 5 (right figure). As a control, CBB (Coomassie Brilliant Blue) staining was performed (left figure). The results of staining a mouse testis section with Reactive Black 5 are shown.

EMBODIMENT OF THE INVENTION Hereinafter, the form for implementing this invention is demonstrated.
The first embodiment is a method of visualizing a sperm head by staining the sperm head with a compound represented by the following formula (I) (hereinafter also referred to as "visualization method according to this embodiment"). do).
The compound represented by formula (I) is a reactive pigment, and is generally often used as a dye. In this specification, for example, "compound of formula (I)", "dye compound of formula (I)", etc. are also described.
In the above formula (I), R ₁ is the same or different 1 to 4 substituents on the benzene ring, each independently representing a hydrogen atom or a lower alkyl group, and R ₂ is the same or different on the benzene ring. One to four substituents each independently represent a hydrogen atom or a lower alkyl group, R ₃ represents a hydrogen atom or a lower alkyl group, and R ₄ represents a hydrogen atom or a lower alkyl group. Here, the "lower alkyl group" may be any substituent having 1 to 10 carbon atoms, preferably 1 to 5 carbon atoms, and having a linear, branched, cyclic, or combination thereof structure.
In this embodiment, _a particularly preferable compound among the compounds _of formula (I) is Reactive black _{5 (} C ₂₆ H ₂₁ N ₅ Na ₄ O ₁₉ S ₆ ; 4-amino-5-hydroxy-3,6-bis[[4-[[2-(sodiosulfoxy)ethyl]sulfonyl]phenyl]azo]-2,7- CAS Number: 17095-24-8).

Reactive Black 5, which is 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 the sperm head, it stains the sperm head dark blue. Therefore, when Reactive Black 5 is added to a sample containing sperm, the sperm heads are dyed dark blue (blue) and visualized. The visualized sperm head can be clearly confirmed by observing it with a microscope.
It is also possible to photograph the staining state of the sperm head with a camera or the like, obtain an image of the stained sperm head, process the image into electronic information, and quantify the staining intensity. For example, the color of a stained sperm head can be divided into red, green, and blue RGB colors using a bioimaging analysis system, and the stainability of each color can be binarized according to the hue. can.

The second embodiment is a method for detecting sperm, which includes visualizing the sperm head by staining the sperm head with a compound represented by formula (I) (hereinafter referred to as (Also referred to as "detection method according to this embodiment"). That is, this is a method of detecting sperm by visualizing the sperm head using the visualization method according to the first embodiment.
Samples to be used in the detection method according to the present embodiment include semen and semen specimens (e.g., semen smear, purified sperm smear, testicular tissue section, etc.) obtained from animals (humans and non-human animals). It is not limited to these. The detection method according to the present embodiment includes a step of contacting a sample such as a semen specimen with a dye compound of formula (I), but this step is not particularly limited. (e.g., an alkaline solution (e.g., about pH 9 to pH 11, preferably about pH 10)) is added to a semen specimen, etc., and incubated (e.g., left standing) at room temperature for several minutes to an hour. This can be done by doing this. Note that the conditions listed here (alkaline conditions, temperature, standing time, etc.) are just examples, and those skilled in the art will be able to select appropriate conditions by conducting appropriate preliminary experiments. can. After bringing the semen specimen into contact with the pigment compound of formula (I) and incubating it, remove the excess pigment compound of formula (I) by washing, etc., and then observe with a microscope etc. for the presence or absence of stained sperm heads. I do.
After treating the sample with the dye compound of formula (I), the heads of spermatozoa, if present, can be stained and visualized and detected in the sample.

In the detection method according to the present embodiment, a person skilled in the art can appropriately select the concentration of the dye compound of formula (I) that is brought into contact with the sample by conducting preliminary experiments. For example, when the dye compound of formula (I) is an aqueous solution of Reactive Black 5, the amount is 0.0001% (w/v) to 0.001% (w/v).

The detection method according to the present embodiment is capable of detecting sperm heads in a short time, simply, and with high sensitivity compared to conventional immunostaining methods, etc. Therefore, the detection method according to the present embodiment is used. This makes it possible to quickly and accurately determine the presence or absence of sperm in a sample from an infertile patient, and also roughly determine the degree of sperm maturity. As a result, it is difficult to accurately judge the selection of treatment methods, such as whether to apply or avoid microdissection testicular sperm extraction (MD-TESE), which is highly physically invasive. It becomes possible.

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 the above formula (I). (hereinafter also referred to as "evaluation method according to this embodiment"). That is, the first embodiment is a method for visualizing sperm heads and evaluating morphological abnormalities of sperm heads.
The sperm head (sperm nucleus) contains small vacuoles at a physiological level even in normal sperm heads. When these vacuoles become multiple or enlarged and occupy more than 1/3 of the nuclear volume, they are called abnormal vacuoles (abnormal sperm). Since the inside of the vacuole is observed as a region with sparse electron density using a transmission electron microscope, it is thought that there is local chromatin condensation failure. Since Reactive Black 5, one of the compounds of formula (I), binds to protamine, vacuoles in which protamine is absent are observed as white holes. Therefore, abnormal vacuoles can be identified even under a normal microscope by reactive black 5 staining.

Although abnormal vacuoles are considered to be a clear morphological abnormality, there is currently no consensus as to whether this morphological abnormality has a functionally adverse effect on pregnancy. However, the intranuclear vacuoles of the spermatozoa are present in many ways from fertilization to the next generation. There are also reports suggesting that it has negative effects on all processes up to (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 morphology of the sperm head using the evaluation method according to the present embodiment, it may be possible to predict the possibility of adverse effects after fertilization such as a decrease in early embryonic development.
In addition, head depression and intranuclear cannon, which are physiological changes often observed in sperm, are difficult to distinguish using low-resolution microscopy. In contrast, the evaluation method according to the present embodiment makes it possible to distinguish between a physiological change in which the sperm head is depressed and an abnormal vacuole.

The fourth embodiment is a method for detecting protamine in a sample, which includes binding a compound represented by the above formula (I) to protamine and visualizing the protamine.
Protamine is a sperm-specific nuclear protein that is thought to be involved in the condensation of the sperm nucleus and its decondensation after entry into the egg. There are 1 to 4 protamines (P1, P2, P3, and P4), and among these, the main components of protamine contained in sperm are P1 and P2. 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 Western blotting methods in which P1 and P2 are detected separately using their respective specific antibodies. Conventional methods for detecting P1 and P2 using Western blotting require complicated steps and are time-consuming and costly to obtain results.

Reactive Black 5 specifically binds to protamine and is therefore useful for detecting protamine. For example, after performing gel electrophoresis on a sample containing protamine (such as a sample extracted from a sperm head), Reactive Black 5 can be bound to protamine by simply immersing the gel directly in Reactive Black 5 solution. . As a result, P1 and P2 are detected as dark blue bands on the gel. The abundance ratio of P1 and P2 can be quantified by quantifying the staining concentration of bands detected by densitometry or the like.
Compared to conventional detection methods using protamine antibodies, time, effort, cost, etc. can be significantly reduced. 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, which can greatly affect the results such as sample misapplication or uneven transfer. It is possible to avoid procedural artifacts and measure the P1/P2 ratio more accurately.

The fifth embodiment is a reagent for visualizing sperm heads or protamine, which contains at least a compound represented by the above formula (I).
The reagent according to the present embodiment may contain the compound of formula (I), such as Reactive Black 5, as well as additives for reagent preparation. The reagent according to the present embodiment is not particularly limited, but may be in the form of a liquid, powder, solid (tablet, tablet, etc.), and if necessary, may contain a pH adjuster, a preservative, etc. It may contain ingredients.
To produce a reagent in solid form, for example, the compound of formula (I) as an active ingredient and an excipient component may be mixed to form a powder, or a binder, a disintegrant, etc. may be added to produce a wet or dry process. It may also be made into granules. Furthermore, in order to manufacture tablets, these powders and granules may be compressed as they are or with the addition of a lubricant.

In the embodiment 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 livestock animals such as cows, pigs, sheep, and horses. It's not something you can do.

When this specification is translated into English and includes the words "a,""an," and "the," the words "a,""an," and "the" refer to the singular as well as the plural, unless the context clearly indicates otherwise. This shall also include those of
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.

1. Experimental method 1-1. Staining of sperm heads with Reactive Black 5 Sperm smears (semen, washed sperm, fractionated sperm, etc.) were taken to the Obstetrics and Gynecology Department of Ichikawa General Hospital.
The specimen was obtained from a male patient who consented to its use. In addition, Reactive Black 5 was purchased from Sigma-Aldrich (Cat# 306452, Dye content≧50%) and dissolved in milli Q to prepare a 0.1% (w/v) storage solution. Before use, the stock solution was diluted 100 times with 0.1 M carbonate-bicarbonate buffer (pH 10).
The area around the specimen on the slide glass was surrounded with a pap pen, an appropriate amount of 0.001% (w/v) Reactive Black 5 was mounted on the area (specimen area), and the area was left standing at room temperature for 10 minutes. Thereafter, excess pigment on the specimen was washed away with water, the specimen was sealed with a cover glass, and then microscopic observation was performed.

1-2. Binarization of stained images Open the TIFF image taken under a microscope with ImageJ and convert it to an 8-bit grayscale image. This image was binarized by changing the upper and lower limits of Threshold and setting values that would allow individual sperm to be distinguished from the background without whiteout or blackout.

1-3. Staining of mouse testis 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, mounted with an appropriate amount of 0.01% (w/v) Reactive Black 5, and left at room temperature for 60 minutes. Thereafter, excess pigment on the specimen was washed away with water, the specimen was sealed with a cover glass, and then microscopic observation was performed.

1-4. Extraction of sperm protamine
After mixing the sperm suspension and the extract at a ratio of 1:2, 1/100 volume of 500 mM DTT was added and mixed, which immediately gelled, so the mixture was left for about 5 minutes.
The preparation liquid used here was prepared as follows.
extract liquid
0.5M Na₂C.O.₃- NaHCO₃ pH 10.0
10 M urea
0.225M Na₂S.O._Four
The extract with the above composition is 0.5M Na₂C.O.₃- NaHCO₃ pH 10.0 (Na in 400 ml of water)₂C.O.₃10.6g, NaHCO₃40 mL, urea 60 g, Na₂S.O._FourIt was prepared by diluting 3.2 g to 100 mL with water.
500mM DTT
500mM DTT
20mM acetate buffer pH 4.7
A 1.0 M acetate buffer (prepared by mixing equal amounts of 60 g (1.049 g/ml, 57 ml)/L acetic acid and 82 g/L sodium acetate) was used to prepare the above DTT stock.

To promote the dissociation of the DNA/protamine complex, 1 volume of a 6.0% benzalkonium chloride 40% ethanol solution (final concentration 1.5%) was added to 3 volumes of the gelled extract, and the mixture was stirred with a vortex mixer. Stirring causes DNA to precipitate into threads and condense. The aggregated DNA was removed by collecting it into a precipitate by centrifugation or by pinching it with tweezers. Then, in order to precipitate protamine, an equal amount of ethanol was added to the obtained extract, stirred with a vortex mixer, and after centrifugation, protamine was recovered as a white precipitate. The supernatant after centrifugation was removed by decantation, and a small amount of 50% ethanol that had accumulated at the bottom was removed with a pipette.

1-5. Electrophoresis of sperm protamine Electrophoresis was performed under acidic conditions to prevent the SH groups of extracted protamine from reassociating. The pI of protamine is 11-12. Protamine is composed of almost a single amino acid, arginine, and is a basic protein, so its charge does not change depending on pH, and its mobility is not affected by pH. As a polyacrylamide gel for electrophoresis, a concentration gel was omitted and only an 18% acrylamide (24:1) separation gel was prepared.
Sample buffer was added to the protamine precipitate. The composition of the sample buffer is as follows.
sample buffer
7.0M Urea
0.1 M _Na2CO3 - _NaHCO3 , _pH 10.0
Before use: Add 1/100 volume of 500mM DTT After adding sample buffer to the protamine precipitate, stir with a vortex mixer to dissolve protamine. If insoluble matter was generated, it was removed by centrifugation.

The compositions of the acrylamide gel for separation and the buffer used for electrophoresis are shown below.
Mixed liquid for gel preparation
77.76 g of acrylamide and 3.24 g of N, N'-methylenebis(acrylamide) were dissolved in 300 mL of water.
separation gel buffer
Solid NaOH was added to 114 mL of glacial acetic acid to adjust the pH to 3.8-4.0, and the volume was diluted to 1000 mL with water to prepare a 2.0 M acetate buffer.
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), TEMED (N, Water was added to N,N',N'-Tetramethylethylene-Diamine (90 μL) to make a total volume of 45 mL, and after mixing, the mixture was allowed to stand at 36°C for 30 minutes or more.
running buffer
(1) Lower running buffer
0.3 M acetate buffer pH 4.8 was used.
(2) Upper running buffer
Glycine (30 g) and glacial acetic acid (0.3 ml) were diluted to 2 L with water.

In this example, a 1.5 mm thick gel was used. Note that the electrophoresis tank is preferably cooled with an ice bath.
Electrophoresis conditions
First stage (concentration) constant current 60mA
Second stage (separation) constant current 60mA
Electrophoresis was performed under the above conditions for approximately 3.0 hours.
Note that the conditions for electrophoresis can be set by those skilled in the art by conducting 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 by electrophoresis at 60 mA (CC) for about 30-40 minutes, has entered the gel (first step (concentration)) ), stop the electrophoresis once. Remove 1/10 volume of the upper running buffer and add 20 ml of 1.0 M sodium acetate equal to the volume removed to make the upper running buffer a 0.10 M sodium acetate solution. Then, electrophoresis is performed at 60 mA (CC) (second step (separation)).

1-6. Staining of protamine with Reactive Black 5
Preparation of staining solution
0.2% _Reactive Black 5, 0.1 M _Na2CO3 - _NaHCO3 pH 10.0 was prepared as a staining solution stock.
At the time of use, the pH was adjusted to 10.7 by adding NaOH dropwise to this stock solution. An acrylamide gel on which protamine was electrophoresed was immersed in the pH-adjusted staining solution, allowed to stand for about 30-40 minutes at room temperature, and then immersed in water and left to stand overnight to decolorize it. Since Reactive Black 5 has high hydrophilicity, it is sufficient to decolor it by immersing it in water, but if decoloring is to be performed in a shorter time, it may be immersed in, for example, 10% isopropanol/water.
CBB staining performed for comparison was performed in 0.2% CBB (50% methanol/water). Specifically, an acrylamide gel was sandwiched between mesh sheets, immersed in the above CBB staining solution, and left to stand for 30 to 40 minutes. Thereafter, the acrylamide gel sandwiched between mesh sheets was immersed in water, an appropriate amount of a weakly basic anion exchange resin was added, and the gel was gently stirred with a stirrer to decolorize it overnight.

2. Result 2-1. Confirmation of staining property of Reactive Black 5 using semen specimen Conventionally, when no cells with tails (sperm) are observed under a phase contrast microscope, azoospermia is diagnosed and the semen is then centrifuged and concentrated. The resulting sediment was observed. Furthermore, methods of tissue staining such as Papanicolaou and Giemsa staining have been attempted. However, under a phase contrast microscope, the transparent particles overlapped, making it difficult to observe the sperm. Furthermore, when Papanicolaou staining, Giemsa staining, etc., all the cells (particles) were colored, making observation difficult in this case as well. Furthermore, the most important problem with the conventional method is that it is impossible to observe sperm cells that arrest during the spermatogenesis process, that is, sperm cells that have stopped forming before obvious tail formation occurs.
The present invention enables specific staining of protamine, and when a concentrated sample is stained with Reactive Black 5, unstained particles are observed (Figure 1 left and Figure 2), and only cells containing protamine are stained blue. (Fig. 1 right and Fig. 2), making it possible to clearly observe cells containing protamine.
The above results suggest that there are cases in which the physically invasive MD-TESE can be avoided among cases that were previously judged to have azoospermia.

On the other hand, in some of the cases studied, although there were sperm (like cells) stained with Reactive Black 5, their morphology was so severely damaged that they could not be recognized as sperm at first glance (Fig. 1 right). That is, sperm-like cells that contain protamine but cannot be recognized as sperm under normal microscopy and protamine-containing degenerated sperm cells called "residual bodies" were detected by reactive black 5 staining.
Although it is not appropriate to use the abnormal spermatozoa described above for microinsemination, the fact that they contain protamine indicates that the defect in spermatogenesis has occurred at least after the incorporation of protamine. In other words, in these cases, it is suggested that there is a high possibility that sperm cells are present in the testes, although they are relatively immature, and this could be a promising application example of MD-TESE. Conversely, in cases where there are no Reactive Black 5-positive sperm in the semen smear, there is a high possibility that no sperm will be obtained even if MD-TESE is performed.
Furthermore, the results shown in Figures 1 and 2 suggest that Reactive Black 5 staining is effective even in sperm detection under MD-TESE. That is, under MD-TESE, the method of visualizing sperm using Reactive Black 5 staining can help quickly determine the presence or absence of sperm (like cells) that can be used for microinsemination.

When sperm were stained with Reactive Black 5 at different concentrations, it was found that the contrast of the stained image was clearer when stained with a higher concentration, but the color fading in the vacuole area (the white area) was worse. (Figure 3). Therefore, it is desirable to appropriately select the density of the reactive black 5 used depending on the purpose of observation.

Next, images of sperm stained with Reactive Blue 2 (0.05%) and Reactive Black 5 (0.001%) were compared (Figure 4). As a result, it was confirmed that the contrast of the stained image at low density was clearer when dyed with Reactive Black 5.
Next, the images of sperm stained with 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 and binarized so that almost all of the sperm that could be confirmed by dye staining remained without blackout or whiteout. This was counted using an automatic cell recognition function, and the difference from the visually observed sperm count was compared. As a result, with Reactive Blue 2, more stained somatic cells were included in the count, and the dissociation was 9.17 times higher than the visually observed sperm count. On the other hand, since Reactive Black 5 showed almost no staining in somatic cells, the difference from the visually observed sperm count was only 2.64 times. The discrepancy from the actual number in Reactive Black 5 staining is due to the fact that the staining density within one sperm was counted as multiple sperm during automatic measurement, and this is because the definition (minimum to maximum area) of counting as "one cell" was set. I think it is possible to get closer to real numbers by doing this.

2-2. Visualization of abnormal sperm head morphology using Reactive Black 5 Even in normal sperm, there are small vacuoles at a physiological level in the sperm head, but these become numerous or enlarged and become more than 1/3 of the sperm nucleus volume. Those that occupy this area are called abnormal vacuoles (abnormal sperm). Since the inside of the vacuole is observed as a region with sparse electron density using a transmission electron microscope, it is thought that there is local chromatin condensation failure. Vacuole regions where protamine does not bind and chromatin aggregation failure occurs are not stained with Reactive Black 5 and are observed as white. In Figure 6, in the sperm head treated with 1 mM DTT to loosen the protamine-protamine cross-link, the vacuoles disappear and the stainability with Reactive Black 5 decreases (see Figure 6, "1 mM DTT process"). When sperm heads were stained with Reactive Black 5 without DTT treatment, white areas were observed (no treatment in Figure 6). This white area is thought to be due to the absence of protamine, resulting in chromatin aggregation failure and vacuolation. In fact, this hypothesis is based on the fact that in the transmission electron microscopy images of head-vacuolated spermatozoa, the vacuoles are filled with chromatin-like structures with low electron density rather than condensed protamine (TEM in Figure 7), and that In the immunostaining image of the vacuolar spermatozoa, the vacuole area (arrow) has sparse DNA (top right of Figure 7) and protamine 2 (PRM2) (bottom right of Figure 7) compared to the surrounding area, while histone H3 is condensed. This is also supported by the fact that (bottom left of Figure 7).
From the above, the vacuoles present in the sperm head, including abnormal vacuoles, come out white when Reactive Black 5 staining is performed, and can be detected even with a normal microscope (Figure 6). Furthermore, FIG. 8 shows that abnormalities in the shape of sperm heads can also be clearly detected by Reactive Black 5 staining.

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

2-4. Utilization of Reactive Black 5 staining for non-human semen analysis Protamine is a protein found in the sperm of most animals, from fish to higher mammals. It can be widely applied to animal sperm research and industry. So far, we have confirmed that Reactive Black 5 does indeed bind to salmon and herring (fish), mouse, and pig protamine. As an example, FIG. 10 shows the results of visualizing intratesticular sperm of a mouse using Reactive Black 5 staining.

The present invention, as an alternative method of detecting an immune reaction using an anti-protamine antibody, can be widely applied not only to medical applications for male infertility, but also to the detection of protamine, including applications to the livestock industry and basic research. Therefore, the present invention is expected to be used in a wide range of fields such as the livestock field and the medical field.

Claims (9)

A method of visualizing a sperm head by staining the sperm head with a compound represented by the following formula (I).
(In formula (I), R ₁ is the same or different 1 to 4 substituents on the benzene ring, each independently representing a hydrogen atom or a lower alkyl group, and R ₂ is the same or different on the benzene ring. 1 to 4 substituents, each independently representing a hydrogen atom or a lower alkyl group, R ₃ represents a hydrogen atom or a lower alkyl group, R ₄ represents a hydrogen atom or a lower alkyl group) 2. The method of claim 1, wherein _R1 , _R2 , _R3 and _R4 are hydrogen. A method for detecting sperm, the method comprising visualizing the sperm head by the method according to claim 1 or 2. A method for evaluating morphological abnormalities in sperm heads, the method comprising visualizing the sperm heads by the method according to claim 1 or 2. 5. The method according to claim 4, wherein the morphological abnormality is an abnormal vacuole. A method for detecting protamine in a sample, the method comprising binding a compound represented by the following formula (I) to protamine and visualizing the protamine.
(In formula (I), R ₁ is the same or different 1 to 4 substituents on the benzene ring, each independently representing a hydrogen atom or a lower alkyl group, and R ₂ is the same or different on the benzene ring. 1 to 4 substituents, each independently representing a hydrogen atom or a lower alkyl group, R ₃ represents a hydrogen atom or a lower alkyl group, R ₄ represents a hydrogen atom or a lower alkyl group) 7. The method of claim 6, wherein _R1 , _R2 , _R3 and _R4 are hydrogen. A reagent for visualizing sperm heads or protamine, which comprises at least a compound represented by the following formula (I).
(In formula (I), R ₁ is the same or different 1 to 4 substituents on the benzene ring, each independently representing a hydrogen atom or a lower alkyl group, and R ₂ is the same or different on the benzene ring. 1 to 4 substituents, each independently representing a hydrogen atom or a lower alkyl group, R ₃ represents a hydrogen atom or a lower alkyl group, R ₄ represents a hydrogen atom or a lower alkyl group) 9. The reagent according to claim 8, wherein _R1 , _R2 , _R3 and _R4 are hydrogen.