CN103454416A - Method for detecting neuraminidase influencing sperm functions - Google Patents
Method for detecting neuraminidase influencing sperm functions Download PDFInfo
- Publication number
- CN103454416A CN103454416A CN2013104318470A CN201310431847A CN103454416A CN 103454416 A CN103454416 A CN 103454416A CN 2013104318470 A CN2013104318470 A CN 2013104318470A CN 201310431847 A CN201310431847 A CN 201310431847A CN 103454416 A CN103454416 A CN 103454416A
- Authority
- CN
- China
- Prior art keywords
- sperm
- sialidase
- detection method
- capacitation
- expression
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a method for detecting neuraminidase influencing sperm functions. The method is a novel laboratory detecting method for assessing quality and functions of a sperm and can provide detection and clinical consultations for male infertility patients without clear clinical reasons for infertility. The method is characterized in that expressions of neuraminidase in the sperm are visually quantized through single sperm fluorescence strength analysis or are visually observed by means of fluorescence imaging under a microscope, and if the neuraminidase is highly expressed in the sperm, capacitation of the sperm is achieved through external capacitation liquid, and neuraminidase activity is detected in a fluorescence method.
Description
Technical field
The invention belongs to medical detection technology, be specifically related to a kind of sialidase detection method that affects sperm function.
Background technology
Along with the development of society's industry, the total incidence of infertile couples obviously raises.Within 2004, European genesiology can be added up: the couple at child-bearing age can not conceived person account for 25% in 1 year, wherein 15% sought treatment, and bridegroom's or husband's side factor causes and sterilely accounts for 50%.The cause of disease of male sterility has sex dysfunction (1.7%), varicocele (12. 3%), genital tract infection (6.6%), congenital dysplasia (2.1%) the acquired disease day after tomorrow (2.6%), endocrine disturbance (0.6%), immunity factor (3.1%), other abnormal (3%), but the patient up to 60% ~ 75% can not find reason, be called idiopathic male infertility, it is abnormal that they only show as the sperm qualities such as few essence, weak essence or teratozoospermia.The male sterility of unknown cause may cause due to many factors, as the long-term stress environmental factor causes endocrine disturbance, active oxygen and gene defect etc.
For many years, traditional seminal fluid conventional analysis is for judging the most basic clinical indices of male fertility always.Still unclear to most male sterility patients' real sterile reason clinically.Especially clinically nearly 1/3rd infertile patients, the male sex's conventional semen analysis result is all normal and approach normal.Clinically this class patient is divided into to Unexplained infertility.Therefore, for a long time, there is very large difficulty in the clinical diagnosis to male sterility.Topmost reason is the quantity that sperm is only measured in conventional semen analysis, viability, activity ratio and form.These indexs can only reflect the most basic semen quality, can not reflect all sperm functions, such as, the maturation of sperm nucleus and DNA damage, sperm and human oocyte zona pellucida association reaction with penetrate, acrosome situation and reaction and with the vitellinae membrana binding ability etc.Therefore, special sperm function tests need to be set up and above these functions could be measured.Capacitation is smart ovum in conjunction with the necessary condition that forms embryonated egg, although the more existing progress of the correlative study of capacitation, but still have much still undefined problem.
In clinical position, the sterile male patient of a part former or secondary utilizes existing conventional semen quality inspection item prompting sperm and semen quality without extremely.Sperm detection means commonly used is CASA (the area of computer aided sperm is analyzed Computer aided of semen analysis) at present, and AsAb etc.The most widely used CASA technology and some morphologic coherent detections now, mainly can estimate the mobility of sperm and the sperm ratio of living, thereby infer that it enters the fertility of female genital tract, and rely on current sperm function appraisement system, there is certain defect in evaluation to sperm quality and function, and is difficult to provide corresponding on to patient.In fact, this is only an aspect estimating sperm function, and we should consider influential sperm fecundity also does not have a revealed factor more.
As document " sialic acid that the sialidase of mammal sperm self participates in the capacitation process comes off " (Sialidases on mammalian sperm mediate deciduous sialylation during capacitation; " The Journal of Biological Chemistry ", 2012 Nov 2; 287 (45): 38073-9), reported first the sialidase on the sperm membrane (NEU1/3) be the new factor that affects the sperm fecundity.Sialic acid (sialic acid) mode with single glycan molecule in mouse and people's In-vitro Capacitation process comes off from the cell membrane of sperm, and meanwhile, sialidase on sperm membrane (NEU1/3) may come off due to the mobility of sperm membrane participate in to shear and cause single sialic acid molecule and come off, the activity that document has confirmed to suppress NEU3 can reduce the expression of phosphorylation ErK in the capacitation process of sperm, and the low expression of sterile patient's sperm these the two kinds of enzymes of a small amount of unknown cause.
Can be used for estimating sperm function although document has proposed sialidase, and disclose the detection method of sialidase in mouse and a small amount of freezing sperm of people.But document only discloses the detection of expression of sialidase in sperm, the activity of sialidase is not detected to the further report of work; And disclosed detection of expression method can't detect the sialidase in fresh spermatozoa smoothly, can't be applicable to clinical practice.
Summary of the invention
The present invention, in order to solve the problems of the technologies described above, has proposed a kind of sialidase detection method that affects sperm function.The method is a kind of new assessment sperm quality and the laboratory detection method of function, can be clinical agnogenic male sterility patient detection and on are provided.
For achieving the above object, the present invention adopts following technical scheme:
Affect the sialidase detection method of sperm function, it is characterized in that: the first step, quantize intuitively the expression of sialidase in sperm by the analysis of monosperm fluorescence intensity, or utilize fluorescence imaging under microscope to observe intuitively the expression of sialidase in sperm; If sialidase is high expressed in sperm, carry out second step, utilize In-vitro Capacitation liquid to make capacitation, and use the Fluorometric assay sialidase activity.
Described In-vitro Capacitation liquid is HSA (human serum albumins) 5mg/ml HTF(HOF).
The described first step, according to following step, carry out respectively the detection of expression of sperm sialidase NEU1 and NEU3:
A collects fresh liquefaction semen sample, and low-speed centrifugal is isolated sperm, the PBS(phosphate buffer) the washing sperm, fully remove residual refining composition;
B counts sperm quantity, puts 20 ~ 30 min on ice, for braking sperm, is convenient to follow-up operation;
C employing 0.1%
tritonthe X-100(Triton X-100) sperm is carried out to pre-service, after PBS washing sperm, the PFA(paraformaldehyde with 3%) again sperm is carried out to pre-service;
D, when detecting NEU1, adopts 1% BSA(bovine serum albumin(BSA))-PBS solution sealed; Perhaps, when detecting NEU3, adopt methyl alcohol to fix;
E adopts anti-human NEU1 antibody incubation 1 ~ 3 h of 1ug/ul, fully guarantees the Ag-Ab binding time, and PBS washs sperm;
At normal temperatures, sperm is hatched 1 ~ 2 h to F in the conventional fluorescence two of 1:1000 is anti-;
G adopts flow cytometer detection or fluorescent microscope to make the cell smear of sperm, observes the expression of sialidase in sperm.
Described second step, concrete operation step is as follows:
The fresh liquefaction semen sample of A is collected, and the low-speed centrifugal separated sperm keeps sperm integrality and vigor;
B adopts upper reaches method, the CO 5%
2cultivate sperm in cell culture incubator, results upstream sperm, further obtain the good sperm of vigor, adopt BWW(Biggers, Whitten, and Whittinghamreedit) nutrient solution washing sperm, fully remove the compositions such as albumen residual in refining, avoids increasing the non-specific fluorescence background and remove " decapacitation factor ".
Because sperm after death can discharge sialidase, interference experiment increases non-specific expression, adopts upper reaches method can guarantee the capacitation that vigor is good, avoids Necrospermia.
C counts sperm quantity, with In-vitro Capacitation liquid HAS 5mg/ml HTF, the CO 5%
2hatch sperm 3 ~ 4 h in cell culture incubator;
The solution centrifugal that D first will obtain through aforesaid operations separates, and the supernatant of collecting centrifuging again, collect supernatant, fully removes the visible components such as sperm, avoids increasing the non-specific fluorescence background;
E is immediately by the Fluorometric assay sialidase activity of the supernatant after capacitation.
The A step of described first and second step, with speed low-speed centrifugal 5 ~ 6min of 500 ~ 600 g/min, to guarantee that sperm morphology is complete, separate with refining.
The B step of described second step, adopt upper reaches method under 36 ~ 37 ℃, the CO 5%
2cultivate 10 ~ 30min in cell culture incubator.
The C step of described second step, the counting sperm quantity, be adjusted into 1 * 10
7more than/ml, both guaranteed enough physiological In-vitro Capacitation environment that approaches, and made again its sialidase activity be detected;
The C step of described second step, use In-vitro Capacitation liquid HAS 5mg/ml HTF under 36 ~ 37 ℃, 5% CO
2hatch 3 ~ 4 h in cell culture incubator, reaction volume 100 ~ 200ul.
Because the relative bacterium of the enzymatic activity of sperm is very low, so the sensitivity in order to guarantee, must control reaction volume is 100 ~ 200ul, has not only guaranteed capacitation but also has guaranteed the concentration of surveying of enzyme.
The D step of described second step, first with speed low-speed centrifugal 5 ~ 6min of 500 ~ 600 g/min, then, with speed low-speed centrifugal 10 ~ 15min of 5000 ~ 6000g/min, fully remove the visible components such as sperm.
Beneficial effect of the present invention is:
1, the invention provides a kind of sialidase detection method that affects sperm function, is new assessment sperm quality and the laboratory detection method of function, to the patient without wound, for the primary sterility patient provides more detection index and clinical interpretation.
2, the present invention detects and assesses the sialidase in sperm by fluorescence method, the sialic acid that has participated in sperm surface due to the sialidase come off from sperm in the capacitation process is sheared, because detecting the active theoretical foundation of sialidase is to remove the sialic acid compound of shear zone fluorescence with sialidase, if any activity, band fluorophor separation energy produces fluorescence, so can detect and assess its activity.Therefore, this method is the high-sensitivity detection method of the sialidase activity needs of sperm.
3, the present invention, when detecting the expression of sialidase in sperm, puts sperm 20 ~ 30 min on ice, for braking sperm, makes fresh liquefaction sperm motility static, is convenient to follow-up operation.
4, the present invention, when detecting sialidase activity, adopts upper reaches method to obtain the good sperm of vigor, and because sperm after death can discharge sialidase, interference experiment increases non-specific expression, and upper reaches method can guarantee the capacitation that vigor is good, avoids Necrospermia; Simultaneously, adopt BWW nutrient solution washing sperm, fully remove the compositions such as albumen residual in refining, avoid increasing the non-specific fluorescence background and remove " decapacitation factor ", guaranteed the accuracy of experiment.
5, the present invention, when detecting sialidase activity, takes the method for twice centrifuging, fully removes the visible components such as sperm, avoids increasing the non-specific fluorescence background.And the present invention strictly controls centrifugal speed, first with speed low-speed centrifugal 5 ~ 6min of 500 ~ 600 g/min, then, with speed low-speed centrifugal 10 ~ 15min of 5000 ~ 6000g/min, further fully remove the visible components such as sperm, avoid bringing interference for detection.
6, the present invention, when gathering sperm, with speed low-speed centrifugal 5 ~ 6min of 500 ~ 600 g/min, to guarantee that sperm morphology is complete, separates with refining fully.
7, the present invention is when detecting sialidase activity, and the counting sperm quantity, be adjusted into 1 * 10
7more than/ml, both guaranteed enough physiological In-vitro Capacitation environment that approaches, and made again its sialidase activity be detected.
8, the present invention, when detecting sialidase activity, uses In-vitro Capacitation liquid HAS 5mg/ml HTF under 36 ~ 37 ℃, 5% CO
2hatch 3 ~ 4 h in cell culture incubator, the control reaction volume is 100 ~ 200ul.Because the relative bacterium of the enzymatic activity of sperm is very low, so the sensitivity in order guaranteeing must to be controlled reaction volume, had not only guaranteed capacitation but also guaranteed the concentration of surveying of enzyme.
The accompanying drawing explanation
Fig. 1 is the expression of sialidase (NEU1/3) unknown cause male sterility patient.
Embodiment
Below in conjunction with embodiment, essentiality content of the present invention is described in further detail.
Embodiment 1
Affect the sialidase detection method of sperm function, it is characterized in that: the first step, quantize intuitively the expression of sialidase in sperm by the analysis of monosperm fluorescence intensity, or utilize fluorescence imaging under microscope to observe intuitively the expression of sialidase in sperm; If sialidase is high expressed in sperm, carry out second step, utilize In-vitro Capacitation liquid to make capacitation, and use the Fluorometric assay sialidase activity.
Embodiment 2
Affect the sialidase detection method of sperm function, it is characterized in that: the first step, quantize intuitively the expression of sialidase in sperm by the analysis of monosperm fluorescence intensity, or utilize fluorescence imaging under microscope to observe intuitively the expression of sialidase in sperm; If sialidase is high expressed in sperm, carry out second step, utilize In-vitro Capacitation liquid to make capacitation, and use the Fluorometric assay sialidase activity.
Described In-vitro Capacitation liquid is HSA 5mg/ml HTF.
Embodiment 3
The embodiment of the present embodiment is substantially the same manner as Example 1, on this basis:
According to following step, carry out the detection of expression of sperm sialidase NEU1:
A collects fresh liquefaction semen sample, and centrifuging goes out sperm, PBS washing sperm 2 times;
B counts sperm quantity, puts 20 min on ice;
C employing 0.1%
tritonx-100 carries out pre-service to sperm, PBS washing sperm 2 times, and the PFA with 3% carries out pre-service to sperm again;
The BSA-PBS solution of D employing 1% is sealed;
E adopts anti-human NEU1 antibody incubation 1 h of 1ug/ul, PBS washing sperm 2 times;
At normal temperatures, sperm is hatched 1 h to F in the conventional fluorescence two of 1:1000 is anti-;
G adopts flow cytometer detection or fluorescent microscope to make the cell smear of sperm, observes the expression of sialidase in sperm.
Embodiment 4
The embodiment of the present embodiment is substantially the same manner as Example 1, on this basis:
According to following step, carry out the detection of expression of sperm sialidase NEU3:
A collects fresh liquefaction semen sample, and centrifuging goes out sperm, PBS washing sperm 2 times;
B counts sperm quantity, puts 30 min on ice;
C employing 0.1%
tritonx-100 carries out pre-service to sperm, PBS washing sperm 2 times, and the PFA with 3% carries out pre-service to sperm again;
D adopts methyl alcohol to fix;
E adopts anti-human NEU1 antibody incubation 3 h of 1ug/ul, PBS washing sperm 2 times;
At normal temperatures, sperm is hatched 2 h to F in the conventional fluorescence two of 1:1000 is anti-;
G adopts flow cytometer detection or fluorescent microscope to make the cell smear of sperm, observes the expression of sialidase in sperm.
Embodiment 5
The embodiment of the present embodiment is substantially the same manner as Example 1, on this basis:
According to following step, detect sialidase activity:
A collects fresh liquefaction semen sample, centrifuging sperm;
B adopts upper reaches method, the CO 5%
2cultivate sperm in cell culture incubator, results upstream sperm, adopt BWW nutrient solution washing sperm.
C counts sperm quantity, with In-vitro Capacitation liquid HSA 5mg/ml HTF, the CO 5%
2hatch sperm 3h in cell culture incubator;
The solution centrifugal that D first will obtain through aforesaid operations separates, and the supernatant of collecting centrifuging again, collect supernatant;
E is immediately by the Fluorometric assay sialidase activity of the supernatant after capacitation.
Embodiment 6
The embodiment of the present embodiment is substantially the same manner as Example 1, on this basis:
According to following step, detect sialidase activity:
A collects fresh liquefaction semen sample, centrifuging sperm;
B adopts upper reaches method under 36 ℃, the CO 5%
2cultivate 10 min in cell culture incubator, results upstream sperm, adopt BWW nutrient solution washing sperm 3 times.
C counts sperm quantity, with In-vitro Capacitation liquid HSA 5mg/ml HTF, the CO 5%
2hatch sperm 4 h in cell culture incubator;
The solution centrifugal that D first will obtain through aforesaid operations separates, and the supernatant of collecting centrifuging again, collect supernatant;
E detects sialidase activity by the supernatant after capacitation with the fluorescence blue laws immediately.
Use the sialidase detection kit (Fluorometric-Blue) of ABCAM (ab138888), detect the sialidase activity in the capacitation supernatant.
Adopt the fluorescence microplate reader of Ex/Em=~ 320/ ~ 450 nm to detect, press the positive control typical curve, calculate the sialic acid activity, unit is: uUAUS/1 * 10
6sperm (micro-unit/1 * 10
6sperm).
Embodiment 7
The first step, according to following step, carry out the detection of expression of sperm sialidase NEU1:
A collects fresh liquefaction semen sample, and centrifuging goes out sperm, with the speed low-speed centrifugal 6min of 500 g/min, and PBS washing sperm 3 times;
B counts sperm quantity, puts 25 min on ice;
C employing 0.1%
tritonx-100 carries out pre-service to sperm, PBS washing sperm 3 times, and the PFA with 3% carries out pre-service to sperm again;
The BSA-PBS solution of D employing 1% is sealed;
E adopts the anti-human NEU1 antibody incubation 2h of 1ug/ul, PBS washing sperm 3 times;
At normal temperatures, sperm is hatched 1.5 h to F in the conventional fluorescence two of 1:1000 is anti-;
G adopts flow cytometer detection or fluorescent microscope to make the cell smear of sperm, observes the expression of sialidase in sperm.
According to following step, carry out the detection of expression of sperm sialidase NEU3 again:
A collects fresh liquefaction semen sample, and centrifuging goes out sperm, with the speed low-speed centrifugal 6min of 500 g/min, and PBS washing sperm 3 times;
B counts sperm quantity, puts 25 min on ice;
C employing 0.1%
tritonx-100 carries out pre-service to sperm, PBS washing sperm 3 times, and the PFA with 3% carries out pre-service to sperm again;
D adopts methyl alcohol to fix;
E adopts the anti-human NEU1 antibody incubation 2h of 1ug/ul, PBS washing sperm 3 times;
At normal temperatures, sperm is hatched 1.5 h to F in the conventional fluorescence two of 1:1000 is anti-;
G adopts flow cytometer detection or fluorescent microscope to make the cell smear of sperm, observes the expression of sialidase in sperm.
Flow cytometer detects the single sperm fluorescence intensity of post analysis, adopt two kinds of comparative approach: a definitely compares: the sample that detects and antibody homotype contrast (the homotype contrast is the immunoglobulin (Ig) that uses Species origin identical with primary antibodie, identical hypotype, same dose and identical immune globulin bletilla hypotype, for elimination due to antibody is non-specific and Cell binding produces background) b relatively: with more single sperm fluorescence intensity between the batch detection sample.
The present invention utilizes monosperm fluorescence intensity analysis (homotype contrast/contrast in batches) can quantize relatively intuitively the expression of sialidase at sperm; Perhaps utilizing fluorescence imaging under microscope can observe intuitively the expression of sialidase, is a kind of method that the detection method of a kind of imaging directly perceived and Relative quantification is assessed sperm expression sialidase (NEU1/3) so the present invention adopts.
Sialidase NEU1 and the high expressed of sialidase NEU3 in sperm are found in above-mentioned detection, second step, according to following operation detection sialidase activity:
A collects fresh liquefaction semen sample, and the centrifuging sperm, with the speed low-speed centrifugal 6min of 500 g/min;
B adopts upper reaches method under 37 ℃, the CO 5%
2cultivate 30 min in cell culture incubator, results upstream sperm, adopt BWW nutrient solution washing sperm 3 times.
C counts sperm quantity, with In-vitro Capacitation liquid HSA 5mg/ml HTF, the CO 5%
2hatch sperm 3.5h in cell culture incubator;
The solution centrifugal that D first will obtain through aforesaid operations separates, and the supernatant of collecting centrifuging again, collect supernatant;
E is immediately by the Fluorometric assay sialidase activity of the supernatant after capacitation.
Embodiment 8
The present embodiment is substantially the same manner as Example 7, on this basis:
According to following operation detection sialidase activity:
A collects fresh liquefaction semen sample, and the centrifuging sperm, with the speed low-speed centrifugal 5min of 600 g/min;
B adopts upper reaches method under 36.5 ℃, the CO 5%
2cultivate 20min in cell culture incubator, results upstream sperm, adopt BWW nutrient solution washing sperm 3 times.
C counts sperm quantity, is adjusted into 1 * 10
7more than/ml, with In-vitro Capacitation liquid HSA 5mg/ml HTF, the CO 5%
2hatch sperm 3.5h in cell culture incubator;
The solution centrifugal that D first will obtain through aforesaid operations separates, and the supernatant of collecting centrifuging again, collect supernatant;
E is immediately by the Fluorometric assay sialidase activity of the supernatant after capacitation.
Embodiment 9
The present embodiment is substantially the same manner as Example 7, on this basis:
According to following operation detection sialidase activity:
A collects fresh liquefaction semen sample, and the centrifuging sperm, with the speed low-speed centrifugal 5.5min of 550 g/min;
B adopts upper reaches method in 36 ℃, the CO 5%
2cultivate 15 min in cell culture incubator, results upstream sperm, adopt BWW nutrient solution washing sperm 3 times.
C counts sperm quantity, is adjusted into 1 * 10
7more than/ml, use In-vitro Capacitation liquid HSA 5mg/ml HTF in 36 ℃, 5% CO
2hatch 3.5 h in cell culture incubator, reaction volume 100ul.
The solution centrifugal that D first will obtain through aforesaid operations separates, with the speed low-speed centrifugal 6min of 500 g/min, and the supernatant of collecting centrifuging again, the speed low-speed centrifugal 15min with 5000g/min, collect supernatant;
E is immediately by the Fluorometric assay sialidase activity of the supernatant after capacitation.
Embodiment 10
The present embodiment is substantially the same manner as Example 7, on this basis:
According to following operation detection sialidase activity:
A collects fresh liquefaction semen sample, and the centrifuging sperm, with the speed low-speed centrifugal 5min of 580g/min;
B adopts upper reaches method under 37 ℃, the CO 5%
2cultivate 2 min in cell culture incubator, results upstream sperm, adopt BWW nutrient solution washing sperm 3 times.
C counts sperm quantity, is adjusted into 1 * 10
7more than/ml, use In-vitro Capacitation liquid HAS 5mg/ml HTF in 37 ℃, 5% CO
2hatch 3.5 h in cell culture incubator, reaction volume 200ul.
The solution centrifugal that D first will obtain through aforesaid operations separates, with speed low-speed centrifugal 5 min of 600 g/min, and the supernatant of collecting centrifuging again, speed low-speed centrifugal 10 min with 6000g/min, collect supernatant;
E is immediately by the Fluorometric assay sialidase activity of the supernatant after capacitation.
Embodiment 11
The present embodiment is substantially the same manner as Example 7, on this basis:
According to following operation detection sialidase activity:
A collects fresh liquefaction semen sample, and the centrifuging sperm, with the speed low-speed centrifugal 5min of 520 g/min;
B adopts upper reaches method under 36 ~ 37 ℃, the CO 5%
2cultivate 18 min in cell culture incubator, results upstream sperm, adopt BWW nutrient solution washing sperm 3 times.
C counts sperm quantity, is adjusted into 1 * 10
7more than/ml, use In-vitro Capacitation liquid HAS 5mg/ml HTF under 36.5 ℃, 5% CO
2hatch 3.5 h in cell culture incubator, reaction volume 150ul.
The solution centrifugal that D first will obtain through aforesaid operations separates, with speed low-speed centrifugal 5 .5min of 540 g/min, and the supernatant of collecting centrifuging again, the speed low-speed centrifugal 12min with 5500g/min, collect supernatant;
E is immediately by the Fluorometric assay sialidase activity of the supernatant after capacitation.
Claims (9)
1. affect the sialidase detection method of sperm function, it is characterized in that: the first step, quantize intuitively the expression of sialidase in sperm by the analysis of monosperm fluorescence intensity, or utilize fluorescence imaging under microscope to observe intuitively the expression of sialidase in sperm; If sialidase is high expressed in sperm, carry out second step, utilize In-vitro Capacitation liquid to make capacitation, and use the Fluorometric assay sialidase activity.
2. the sialidase detection method that affects sperm function according to claim 1, it is characterized in that: described In-vitro Capacitation liquid is HSA 5mg/ml HTF.
3. the sialidase detection method that affects sperm function according to claim 1 is characterized in that: the described first step, according to following step, carry out respectively the detection of expression of sperm sialidase NEU1 and NEU3:
A collects fresh liquefaction semen sample, and centrifuging goes out sperm, and PBS washs sperm;
B counts sperm quantity, puts 20 ~ 30 min on ice;
C employing 0.1%
tritonx-100 carries out pre-service to sperm, and PBS washs sperm, and the PFA with 3% carries out pre-service to sperm again;
D, when detecting NEU1, adopts 1% BSA-PBS solution to be sealed; Perhaps, when detecting NEU3, adopt methyl alcohol to fix;
E adopts anti-human NEU1 antibody incubation 1 ~ 3 h of 1ug/ul, and PBS washs sperm;
At normal temperatures, sperm is hatched 1 ~ 2 h to F in the conventional fluorescence two of 1:1000 is anti-;
G adopts flow cytometer detection or fluorescent microscope to make the cell smear of sperm, observes the expression of sialidase in sperm.
4. the sialidase detection method that affects sperm function according to claim 1 is characterized in that: described second step, and concrete operation step is as follows:
A collects fresh liquefaction semen sample, centrifuging sperm;
B adopts upper reaches method, the CO 5%
2cultivate sperm in cell culture incubator, results upstream sperm, adopt BWW nutrient solution washing sperm;
C counts sperm quantity, with In-vitro Capacitation liquid HSA 5mg/ml HTF, the CO 5%
2hatch sperm 3 ~ 4 h in cell culture incubator;
The solution centrifugal that D first will obtain through aforesaid operations separates, and the supernatant of collecting centrifuging again, collect supernatant;
E is immediately by the Fluorometric assay sialidase activity of the supernatant after capacitation.
5. according to claim 3 or the 4 described sialidase detection methods that affect sperm function, it is characterized in that: described A step, with speed low-speed centrifugal 5 ~ 6min of 500 ~ 600 g/min.
6. the sialidase detection method that affects sperm function according to claim 4 is characterized in that: described B step adopts upper reaches method in 36 ~ 37 ℃, the CO 5%
2cultivate 10 ~ 30 min in cell culture incubator.
7. the sialidase detection method that affects sperm function according to claim 4 is characterized in that: described C step, the counting sperm quantity, be adjusted into 1 * 10
7more than/ml.
8. the sialidase detection method that affects sperm function according to claim 4 is characterized in that: described C step, with In-vitro Capacitation liquid HSA 5mg/ml HTF in 36 ~ 37 ℃, 5% CO
2hatch 3 ~ 4 h in cell culture incubator, reaction volume 100 ~ 200ul.
9. the sialidase detection method that affects sperm function according to claim 4, it is characterized in that: described D step, first with speed low-speed centrifugal 5 ~ 6min of 500 ~ 600 g/min, then with speed low-speed centrifugal 10 ~ 15 min of 5000 ~ 6000g/min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310431847.0A CN103454416B (en) | 2013-09-22 | 2013-09-22 | Affect the sialidase detection method of sperm function |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310431847.0A CN103454416B (en) | 2013-09-22 | 2013-09-22 | Affect the sialidase detection method of sperm function |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103454416A true CN103454416A (en) | 2013-12-18 |
| CN103454416B CN103454416B (en) | 2016-04-13 |
Family
ID=49737017
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310431847.0A Active CN103454416B (en) | 2013-09-22 | 2013-09-22 | Affect the sialidase detection method of sperm function |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103454416B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105695557A (en) * | 2016-03-16 | 2016-06-22 | 四川大学华西第二医院 | Method for evaluating quality of sperm obtaining energy in vitro |
| CN105717307A (en) * | 2016-03-16 | 2016-06-29 | 四川大学华西第二医院 | Kit for evaluating semen quality and use method thereof |
| CN105779568A (en) * | 2016-03-16 | 2016-07-20 | 四川大学华西第二医院 | Reagent kit for quality evaluation of sperm after in vitro capacitation and use method of reagent kit |
| WO2023246254A1 (en) * | 2022-06-21 | 2023-12-28 | 成都思瑞多医疗科技有限公司 | Detection kit of sperm sialidase 1/3 and preparation method therefor, and method for detecting expression level of sperm sialidase 1/3 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5962302A (en) * | 1998-02-20 | 1999-10-05 | Incyte Pharmaceuticals, Inc. | Human N-acetylneuraminate lyase |
| CN101773665A (en) * | 2010-01-20 | 2010-07-14 | 何以丰 | Reproductive protective agent facilitating human fertilization |
-
2013
- 2013-09-22 CN CN201310431847.0A patent/CN103454416B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5962302A (en) * | 1998-02-20 | 1999-10-05 | Incyte Pharmaceuticals, Inc. | Human N-acetylneuraminate lyase |
| CN101773665A (en) * | 2010-01-20 | 2010-07-14 | 何以丰 | Reproductive protective agent facilitating human fertilization |
Non-Patent Citations (1)
| Title |
|---|
| FANG MA ET AL.: "Sialidases on Mammalian Sperm Mediate Deciduous Sialylation during Capacitation", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》, vol. 287, no. 45, 2 November 2012 (2012-11-02), pages 38073 - 38079 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105695557A (en) * | 2016-03-16 | 2016-06-22 | 四川大学华西第二医院 | Method for evaluating quality of sperm obtaining energy in vitro |
| CN105717307A (en) * | 2016-03-16 | 2016-06-29 | 四川大学华西第二医院 | Kit for evaluating semen quality and use method thereof |
| CN105779568A (en) * | 2016-03-16 | 2016-07-20 | 四川大学华西第二医院 | Reagent kit for quality evaluation of sperm after in vitro capacitation and use method of reagent kit |
| WO2017156844A1 (en) * | 2016-03-16 | 2017-09-21 | 四川大学华西第二医院 | Kit for evaluating sperm quality after in vitro capacitation and method of use thereof |
| WO2017156843A1 (en) * | 2016-03-16 | 2017-09-21 | 四川大学华西第二医院 | Kit for evaluating sperm quality and method of use thereof |
| CN105779568B (en) * | 2016-03-16 | 2019-09-24 | 四川大学华西第二医院 | The kit and its application method that sperm quality is assessed after a kind of In-vitro Capacitation |
| WO2023246254A1 (en) * | 2022-06-21 | 2023-12-28 | 成都思瑞多医疗科技有限公司 | Detection kit of sperm sialidase 1/3 and preparation method therefor, and method for detecting expression level of sperm sialidase 1/3 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103454416B (en) | 2016-04-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Rochester | Bisphenol A and human health: a review of the literature | |
| Sobngwi et al. | Ketosis-prone type 2 diabetes mellitus and human herpesvirus 8 infection in sub-saharan africans | |
| CN103454416B (en) | Affect the sialidase detection method of sperm function | |
| CN102037359B (en) | The function evaluation methods of phagocyte | |
| US11591573B2 (en) | Methods of preparing a primary cell sample | |
| WO2017156843A1 (en) | Kit for evaluating sperm quality and method of use thereof | |
| CN103760331B (en) | Refining joint inspection kit | |
| Ricci et al. | Effect of seminal leukocytes on in vitro fertilization and intracytoplasmic sperm injection outcomes | |
| Menkveld | The basic semen analysis | |
| Fernandez-Fuertes et al. | Removal of sialic acid from bull sperm decreases motility and mucus penetration ability but increases zona pellucida binding and polyspermic penetration in vitro | |
| ESHRE Add-ons working group et al. | Good practice recommendations on add-ons in reproductive medicine | |
| CN105671126A (en) | Method for evaluating semen quality | |
| CN109868313A (en) | Application of the HMGA2 gene in Stein-Leventhal syndrome disease | |
| Freund et al. | Addition of formaldehyde releaser imidazolidinyl urea and MOPS buffer to urine samples enables delayed processing for flow cytometric analysis of urinary cells: A simple, two step conservation method of urinary cells for flow cytometry | |
| Iancu et al. | Prolactin Relationship with Fertility and In Vitro Fertilization Outcomes—A Review of the Literature | |
| CN102866153B (en) | Prostatitis joint-detection kit | |
| CN104372065A (en) | Method for detecting quality of assisted reproductive technology by mouse embryo array | |
| CN108152189B (en) | Quantitative detection method for sperm surface clouding protein 1 | |
| Tabibnejad et al. | Assessing ICSI outcome by combining non‐invasive indicators: early time‐lapse morphokinetics and apoptosis in associated cumulus cells among women with the polycystic ovarian syndrome | |
| CN101858917B (en) | Kit for detecting premature rupture of membrane by taking Axl as detection index and preparation method thereof | |
| CN105779568B (en) | The kit and its application method that sperm quality is assessed after a kind of In-vitro Capacitation | |
| CN105695557B (en) | Detect the application method of the kit of the sialidase in external sperm capacitation liquid | |
| CN103760334B (en) | Application and the testing tool of testing tool is prepared using Acrp30 as mark | |
| Levican et al. | Viral shedding dynamics reveals sputum as a reliable and cost-saving specimen for SARS-CoV-2 diagnosis within the first 10 days since symptom onset: a prospective cohort study | |
| RU2402279C1 (en) | Method for prediction of myopia transition to progressive form |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20190308 Address after: 610000 Chengdu City, Sichuan Province, China (Sichuan) Free Trade Pilot Zone, North Tianfu Avenue, Chengdu High-tech Zone, 1480 No. 1 Building A, 3 Floors, 9 Attached No. 3 (Self-numbered) Patentee after: Chengdu Siruido Medical Technology Co., Ltd. Address before: 610000 No. 20, Section 3, Renmin South Road, Wuhou District, Chengdu City, Sichuan Province Patentee before: West China No.2 Hospital, Sichuan University |