CN104560866A - Method for in-vitro sperm capacitation and detection of Kunming dog - Google Patents

Abstract

The invention discloses a method for in-vitro sperm capacitation and detection of a Kunming dog. The method comprises the following steps: (1) preparing sperm washing liquor and capacitation liquor; (2) washing sperm; (3) performing capacitation in vitro; (4) performing capacitation detection; (5) detecting and inducing an acrosomal reaction. On the basis of repeated tests, key factors, such as capacitation time, bovine serum albumin, Ca<2+>, HCO<3-> and optimal combination thereof, influencing in-vitro sperm capacitation of the Kunming dog are ascertained, an efficient in-vitro sperm capacitation system of the Kunming dog is established, CTC (chlortetracycline fluorescence) staining and induction of the acrosomal reaction are combined for detecting sperm capacitation situations, a result is relatively real and credible, and a solid foundation is laid for making a deep research on a molecular mechanism of sperm capacitation of the Kunming dog.

Description

Kunming dog sperm microcytotoxicity and detection method

Technical field

The invention belongs to animals' reproduction biology techniques field, specifically Kunming dog sperm microcytotoxicity and detection method.

Background technology

Although people and mammalian sperm are in just injection or have motor capacity when taking out from cauda epididymis, but can't combine with ovum at once, need to stay for some time in female reproductive tract, a series of physiological acoustic signals occurs and just possesses the ability making fertilizing oocytes, this phenomenon is called " capacitation ".When the sperm of capacitation enter cumulus cell or with ovum zona pellucida in conjunction with time be activated, sperm head front end acrosome breaks, and occurs so-called " acrosomal reaction ", acrosin contained in acrosome is released, support that sperm is through zona pellucida, thus cause sperm-egg fusion, complete fertilization.Large quantity research shows, capacitation is the physiological stage that all mammalian sperms must experience prefecundation.

Early stage capacitation research, adopts the method for capacitation in body, in the jenny reproductive tract of post-coitum, namely takes out sperm for fertilization, so as to analyzing the position of capacitation, time-histories and mechanism thereof.But this research to capacitation in body is usually subject to the restriction of many conditions, such as, sperm always contacts with female reproductive tract secretory product in vivo, and in female reproductive tract, there is the factor affecting capacitation, people are difficult to the internal milieu controlling this complexity, bring great difficulty thus to research capacitation.

For this reason, the research method of In-vitro Capacitation is arisen at the historic moment.Early stage research sperm microcytotoxicity uses the body fluid (as Oviductal Fluid, folliculi liquor and serum) of various animal usually, and the composition relative complex of these body fluid, be difficult to determine which kind of composition participates in bringing out or supporting capacitation.Since Toyoda and Chang report at first adopt a kind of specific chemical culture solution successfully realize in vitro fertilization since, research sperm microcytotoxicity has become relatively easy.Now, In-vitro Capacitation technology can accurately Control release condition, analyzes capacitation and correlation factor thereof one by one, can be disclosed the essence of capacitation in body by the result of study of In-vitro Capacitation.Make most the mankind be benefited, the success of In-vitro Capacitation directly results in the birth of the tube-test baby techniques being widely used in infertility treatment at present.

Although the In-vitro Capacitation of most of animal sperm is again without technology barrier, the molecule mechanism of capacitation still has many problems not yet to illustrate.At present, the animal such as mouse, hamster, ox, dog, sheep, pig and human spermatogoa are used to the research carrying out capacitation molecule mechanism.

Kunming dog is the excellent sizing kind of dog that China uniquely lists world-famous police dog in, it is unique work kind of dog of Chinese independent cultivation, in tracking, differentiate, search poison, search quick-fried, patrol, wait for, warning, fire-fighting, the fields such as detecting a mine play an important role, its germ plasm resource is very valuable, the research carrying out its capacitation is conducive to the development of the in vitro fertilization and artificial propagation techniques of this animal, but have no report at present about the research of Kunming dog capacitation, even the capacitation of other kind of dog, also be detected in that external more early stage fragmentary, not systematic account, the primary condition of not clear and definite dog capacitation.The present invention illustrates the requirement of Kunming dog sperm microcytotoxicity, successfully establishes the In-vitro Capacitation system of this kind of dog sperm, has enriched the content of animal sperm capacitation, for the molecule mechanism of further investigation Kunming dog capacitation has established good basis.

Summary of the invention

The object of the invention is to set up a kind of Kunming dog sperm microcytotoxicity and detection method, is the molecule mechanism establish a firm foundation of further investigation Kunming dog capacitation.

The technical solution used in the present invention is as follows:

Kunming dog sperm microcytotoxicity and detection method, realized by following steps:

(1) preparation of seminal fluid mCCM (W) and capacitation liquid mCCM is washed

By sodium chloride-containing 0.488g, Repone K 0.0356g, potassium primary phosphate 0.0162g, Sodium.alpha.-ketopropionate 0.0028g, Sodium.alpha.-hydroxypropionate 60% aqueous solution 0.338ml, glucose 0.05g, penicillin G sodium salt 10000 IU, Vetstrep 0.005g, phenol red 0.002g in every 100ml solution formula Milli-Q ultrapure water preparation wash seminal fluid mCCM (W), after via hole diameter 0.22 μm of frit by it in 38 DEG C of water bath heat preservations;

By the formula Milli-Q ultrapure water preparation capacitation liquid mCCM of sodium chloride-containing 0.488g, Repone K 0.0356g, calcium chloride 0.0222g, potassium primary phosphate 0.0162g, sodium bicarbonate 0.3159g, Sodium.alpha.-ketopropionate 0.0028g, Sodium.alpha.-hydroxypropionate 60% aqueous solution 0.338ml, glucose 0.05g, bovine serum albumin 0.3g, penicillin G sodium salt 10000 IU, Vetstrep 0.005g, phenol red 0.002g in every 100ml solution, after via hole diameter 0.22 μm of frit, be placed on CO ₂incubator 38 DEG C balance at least 2 hours;

(2) semen washing

The fresh semen 15ml of collection is gone in sterile centrifugation tube, add 10ml mCCM (W) and wash seminal fluid, the centrifugal 5min of 700g, abandon supernatant, add 10ml mCCM (W) again and wash seminal fluid, 700g centrifugal 5min washing once, precipitates that to wash seminal fluid with appropriate mCCM (W) resuspended, makes sperm concentration be 60 × 10 ⁶/ mL;

(3) In-vitro Capacitation

Seminal fluid after above-mentioned washing is divided in 2mL round bottom centrifuge tube, often pipe 300 μ L; Often manage and add 38 DEG C again and thermally equilibratedly contain 2 × (CaCl in advance ₂, NaHCO ₃and BSA) mCCM 300 μ L, centrifuge tube is put CO ₂incubator is cultivated 3 hours in 38 DEG C;

(4) CTC capacitation detects

1., CTC dye liquor preparation: take CTC 2mg, sodium-chlor 38mg, halfcystine 3mg, Tris damping fluid 12mg, adds 5mL Milli-Q ultrapure water, adjusts pH to be 7.8 with 1N NaOH or 1N HCl;

2., dyeing: under lucifuge condition, the slide glass of 38 DEG C of preheatings adds the CTC dye liquor of 10 μ L, then add the sperm suspension of step (2) gained of 10 μ L, 2 μ L 12.5% glutaraldehyde Tris solution are added after 10 seconds, covered gently, is put in wet box, and 4 DEG C of lucifuges are spent the night; Take out slide glass, observe under ultraviolet light, numeration, often organize sum and be no less than 200, staining sperm cells is divided into three types:

" F " type is non-capacitated sperm, and whole sperm head is in brighter yellow-green colour;

" B " type, for capacitation the sperm of acrosomal reaction do not occur, the head acrosome district of sperm is yellow-green colour, and acrosome

Back zone is darker;

" AR " type, for there is the sperm of acrosomal reaction, sperm head back zone is yellow-green colour, and acrosome district is dimmed;

(5) FITC-PSA detects and brings out acrosomal reaction

1., in the sperm of step (3) capacitation 3h, adding calcium ion carrier A 23187 makes its final concentration be 10 μMs, in CO ₂incubator 38 DEG C continues to cultivate 30min;

2., take out the sperm 50 μ L of hatching, add 1mL Tris damping fluid, the centrifugal 5min of 700g, abandons supernatant;

3., precipitation with the 1mL anhydrous methanol of 4 DEG C of precoolings in 4 DEG C of fixing 30s;

4., the centrifugal 5min of 700g, abandon supernatant, add 1mL Tris damping fluid, the centrifugal 5min of 700g;

5., add FITC-PSA, make dye strength be 0.05mg/mL, lucifuge hatches 20min;

6., with PBS wash once, draw 10 μ L on slide glass, with fluorescence microscope, counting, often organizes sum and is no less than 200; Staining sperm cells is divided into two types:

The complete type of acrosome: perforatorium district is in green, and acrosome back zone is dimmed;

Acrosome loss type: perforatorium district and acrosome back zone unstressed configuration, there is a green fluorescence band in sperm stage casing.

Described step (4) and (5) middle Tris damping fluid are 1M Tris-HCl, pH7.8, and its compound method is:

Take 121.14g Tris and add 1LMilli-Q ultrapure water, after fully dissolving, regulate pH to 7.8 with HCl, 4 DEG C of preservations.

In described step (5), FITC-PSA is the pisum sativum agglutinin of marked by fluorescein isothiocyanate.

In described step (4), CTC is the english abbreviation of Isphamycin Chlortetracycline hydrochloride, and Tris is the english abbreviation of Tutofusin tris.

Advantage of the present invention is: on the basis of repetition test, verified affect Kunming dog sperm microcytotoxicity key factor as capacitation time, bovine serum albumin, Ca ²⁺, HCO ₃ ^-and best of breed, establish efficient Kunming dog sperm microcytotoxicity system, being dyeed by CTC and bringing out acrosomal reaction combines to detect capacitation situation, and result is more genuine and believable.

Accompanying drawing explanation

Fig. 1 is the Kunming dog sperm CTC dyeing schematic diagram that the present invention proposes;

Fig. 2 is the Kunming dog sperm FITC-PSA dyeing schematic diagram that the present invention proposes.

Embodiment

Below by way of drawings and the specific embodiments, the present invention is described further.

Embodiment:

1, the preparation of seminal fluid (mCCM (W)) and capacitation liquid (mCCM) is washed

According to the form below formulated capacitation liquid (mCCM)

Composition	Concentration (g/100ml)
		Sodium-chlor	0.488
Repone K	0.0356
		Calcium chloride	0.0222
Potassium primary phosphate	0.0162
		Sodium bicarbonate	0.3159
Sodium.alpha.-ketopropionate	0.0028
		Sodium.alpha.-hydroxypropionate 60% aqueous solution	0.338ml
Glucose	0.05
		Bovine serum albumin	0.3
Penicillin G sodium salt	10000 IU
		Vetstrep	0.005
Phenol red	0.002

Seminal fluid (mCCM (W)) is washed in preparation: not containing CaCl in mCCM (W) i.e. mCCM ₂, NaHCO ₃with BSA tri-kinds of materials.

Wash seminal fluid and capacitation liquid will wash seminal fluid in 38 DEG C of water bath heat preservations after 0.22 μm of frit, capacitation liquid puts CO ₂incubator 38 DEG C balance at least 2 hours.

2, semen washing

The fresh semen of collection is gone in the 15ml centrifuge tube of sterilizing, add 10ml mCCM (W) and wash seminal fluid, the centrifugal 5min of 700g, abandon supernatant, add 10ml mCCM (W) to wash seminal fluid centrifuge washing is once again, precipitate resuspended with appropriate mCCM (W), make sperm concentration be 60 × 10 ⁶/ mL;

3, In-vitro Capacitation

Above-mentioned seminal fluid is divided in 2mL round bottom centrifuge tube, often pipe 300 μ L; Often manage and add 38 DEG C of pre-thermally equilibrated CaCl containing adding double amount by upper table again ₂, NaHCO ₃mCCM 300 μ L with BSA, puts CO by centrifuge tube ₂incubator is cultivated 3 hours in 38 DEG C;

4, CTC capacitation detects

(1) CTC dye liquor preparation: take CTC 2mg, sodium-chlor 38mg, halfcystine 3mg, Tris 12mg, adds 5mL ultrapure water, adjusts pH to be 7.8.

(2) dye: under lucifuge condition, the slide glass of 38 DEG C of preheatings adds the CTC dye liquor of 10 μ L; Add the sperm suspension of 10 μ L again; Add 2 μ L 12.5% glutaraldehyde Tris solution after 10 seconds, covered gently, is put in wet box, and 4 DEG C of lucifuges are spent the night; Take out slide glass, observe under ultraviolet light, count, the present embodiment statistics sum 203 sperms, wherein " B " type+" AR " type sperm is 136, and " F " type sperm is 67, therefore capacitated sperm percentage reaches 67%;

Staining sperm cells is divided into three types, sees accompanying drawing 1:

" F " type is non-capacitated sperm, and whole sperm head is in brighter yellow-green colour;

" B " type, for capacitation the sperm of acrosomal reaction do not occur, the head acrosome district of sperm is yellow-green colour, and acrosome back zone is darker;

" AR " type, for there is the sperm of acrosomal reaction, sperm head back zone is yellow-green colour, and acrosome district is dimmed.

5, the pisum sativum agglutinin of FITC-PSA marked by fluorescein isothiocyanate detects and brings out acrosomal reaction

(1) in the sperm of step 3 capacitation 3h, adding calcium ion carrier A 23187 makes its final concentration be 10 μMs, in CO ₂incubator 38 DEG C continues to cultivate 30min.

(2) take out the sperm 50 μ L of hatching, add 1mLTris damping fluid, the centrifugal 5min of 700g, abandons supernatant;

(3) precipitation with the 1mL anhydrous methanol of 4 DEG C of precoolings in 4 DEG C of fixing 30s;

(4) the centrifugal 5min of 700g, abandons supernatant, adds 1mLTris damping fluid, the centrifugal 5min of 700g.

(5) add FITC-PSA, make dye strength be 0.05mg/mL, lucifuge hatches 20min;

(6) wash once with PBS, draw 10 μ L on slide glass, with fluorescence microscope,

Counting, sum is no less than 200, and the present embodiment has counted 212 sperms, wherein acrosome loss type sperm 193, the complete type sperm of acrosome 19, therefore to bring out acrosomal reaction sperm percentage be 91%.

Staining sperm cells is divided into two types, sees accompanying drawing 2:

The complete type of acrosome: perforatorium district is in green, and acrosome back zone is dimmed.

Acrosome loss type: perforatorium district and acrosome back zone unstressed configuration, there is a green fluorescence band in sperm stage casing.

Correlation analysis: CTC is with to bring out the percentile dependency of acrosomal reaction sperm as follows:

Be 0.993 from the upper table Pearson correlation coefficient that can obtain between two variablees, two-tailed test Probability p value tail 0.000<0.05, therefore Type B sperm percentage and bring out significant correlation between acrosomal reaction sperm percentage.Visible on the basis of capacitation, add acrosomal reaction inductor, can promote capacitated sperm generation acrosomal reaction, further illustrating acrosomal reaction is a kind of physiological response occurred after capacitation, and the index that acrosomal reaction is also capacitation can occur.

Claims (3)

1. Kunming dog sperm microcytotoxicity and detection method, is characterized in that, realizes according to the following steps:

(1) preparation of seminal fluid mCCM (W) and capacitation liquid mCCM is washed

By sodium chloride-containing 0.488g, Repone K 0.0356g, potassium primary phosphate 0.0162g, Sodium.alpha.-ketopropionate 0.0028g, Sodium.alpha.-hydroxypropionate concentration 60% aqueous solution 0.338ml, glucose 0.05g, penicillin G sodium salt 10000 IU, Vetstrep 0.005g, phenol red 0.002g in every 100ml solution formula Milli-Q ultrapure water preparation wash seminal fluid mCCM (W), after via hole diameter 0.22 μm of frit by it in 38 DEG C of water bath heat preservations;

By the formula Milli-Q ultrapure water preparation capacitation liquid mCCM of sodium chloride-containing 0.488g, Repone K 0.0356g, calcium chloride 0.0222g, potassium primary phosphate 0.0162g, sodium bicarbonate 0.3159g, Sodium.alpha.-ketopropionate 0.0028g, Sodium.alpha.-hydroxypropionate 60% aqueous solution 0.338ml, glucose 0.05g, bovine serum albumin 0.3g, penicillin G sodium salt 10000 IU, Vetstrep 0.005g, phenol red 0.002g in every 100ml solution, after via hole diameter 0.22 μm of frit, be placed on CO ₂incubator 38 DEG C balance at least 2 hours;

(2) semen washing

The fresh semen 15ml of collection is gone in the centrifuge tube of sterilizing, add 10ml mCCM (W) and wash seminal fluid, the centrifugal 5min of 700g, abandon supernatant, add 10ml mCCM (W) again and wash seminal fluid, 700g centrifugal 5min washing once, precipitates that to wash seminal fluid with appropriate mCCM (W) resuspended, makes sperm concentration be 60 × 10 ⁶/ mL;

(3) In-vitro Capacitation

Seminal fluid after above-mentioned washing is divided in 2mL round bottom centrifuge tube, often pipe 300 μ L; Often manage and add 38 DEG C again and thermally equilibratedly contain 2 × (CaCl in advance ₂, NaHCO ₃and BSA) mCCM 300 μ L, centrifuge tube is put CO ₂incubator is cultivated 3 hours in 38 DEG C;

(4) CTC capacitation detects

1., CTC dye liquor preparation: take CTC 2mg, sodium-chlor 38mg, halfcystine 3mg, Tris damping fluid 12mg, adds 5mL Milli-Q ultrapure water, adjusts pH to be 7.8 with 1N NaOH or 1N HCl;

2., dyeing: under lucifuge condition, the slide glass of 38 DEG C of preheatings adds the CTC dye liquor of 10 μ L, then add the sperm suspension of step (2) gained of 10 μ L, 2 μ L 12.5% glutaraldehyde Tris solution are added after 10 seconds, covered, is put in wet box, and 4 DEG C of lucifuges are spent the night; Take out slide glass, observe under ultraviolet light, numeration, often organize sum and be no less than 200, staining sperm cells is divided into three types:

" F " type is non-capacitated sperm, and whole sperm head is in brighter yellow-green colour;

" B " type, for capacitation the sperm of acrosomal reaction do not occur, the head acrosome district of sperm is yellow-green colour, and acrosome

Back zone is darker;

" AR " type, for there is the sperm of acrosomal reaction, sperm head back zone is yellow-green colour, and acrosome district is dimmed;

(5) FITC-PSA detects and brings out acrosomal reaction

1., in the sperm of step (3) capacitation 3h, adding calcium ion carrier A 23187 makes its final concentration be 10 μMs, in CO ₂incubator 38 DEG C continues to cultivate 30min;

2., take out the sperm 50 μ L of hatching, add 1mL Tris damping fluid, the centrifugal 5min of 700g, abandons supernatant;

3., precipitation with the 1mL anhydrous methanol of 4 DEG C of precoolings in 4 DEG C of fixing 30s;

4., the centrifugal 5min of 700g, abandon supernatant, add 1mL Tris damping fluid, the centrifugal 5min of 700g;

5., add FITC-PSA, make dye strength be 0.05mg/mL, lucifuge hatches 20min;

6., with PBS wash once, draw 10 μ L on slide glass, with fluorescence microscope, counting, often organizes sum and is no less than 200; Staining sperm cells is divided into two types:

The complete type of acrosome: perforatorium district is in green, and acrosome back zone is dimmed;

Acrosome loss type: perforatorium district and acrosome back zone unstressed configuration, there is a green fluorescence band in sperm stage casing.

2. Kunming according to claim 1 dog sperm microcytotoxicity and detection method, is characterized in that, described step (4) and (5) middle Tris damping fluid are 1M Tris-HCl, pH7.8, and its compound method is:

Take 121.14g Tris and add 1LMilli-Q ultrapure water, after fully dissolving, regulate pH to 7.8 with HCl, 4 DEG C of preservations.

3. Kunming according to claim 1 dog sperm microcytotoxicity and detection method, is characterized in that, in described step (5), FITC-PSA is the pisum sativum agglutinin of marked by fluorescein isothiocyanate.