CN112899220B - HTF liquid drop for improving fertilization capacity of mouse sperm and preparation and use methods thereof - Google Patents
HTF liquid drop for improving fertilization capacity of mouse sperm and preparation and use methods thereof Download PDFInfo
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Abstract
The invention discloses an HTF liquid drop for improving fertilization capacity of mouse sperm, and a preparation method and a use method thereof, wherein the preparation method comprises the following steps: making HTF liquid drops with the volume of 200 mu L/drop in a culture dish, covering the HTF liquid drops with tissue paraffin oil, and then putting the HTF liquid drops into an incubator for balancing; preparing the mouse SERPINA5 artificially synthesized protein powder into a mouse SERPINA5 artificially synthesized protein concentrate by using serum-free HTF, wherein the concentration of the mouse SERPINA5 artificially synthesized protein concentrate is 240-260 mug/mL; and adding the mouse SERPINA5 artificial synthetic protein into the HTF liquid drop for concentration, so that the concentration of the mouse SERPINA5 artificial synthetic protein in the HTF liquid drop is 1-2 mug/mL. The HTF liquid drop for improving the fertilization capacity of the mouse sperm and the preparation and application methods thereof can effectively improve the in-vitro fertilization capacity of the mouse sperm, and provide a reference method for optimizing the sperm quality in the in-vitro capacitation process of the sperm of ART.
Description
Technical Field
The invention relates to the field of sperm in-vitro capacitation, in particular to an HTF liquid drop for improving fertilization capacity of mouse sperm and a preparation method and a use method thereof.
Background
The person who has not been pregnant for more than one year without any contraceptive measure between couples is called infertility, wherein the person is called infertility caused by female factors and the person is called infertility caused by male factors. At present, the incidence rate of infertility in China reaches 15%, the average incidence rate of infertility in developed countries is close to 20%, and the age of the infertility couples tends to be younger. The world health organization considers that infertility will become a further important killer threatening human health, which brings many psychological and physiological injuries to couples and parties, and seriously affects the happiness index of families.
Assisted reproductive technology (Assisted Reproductive Technology, ART) is an important means of treating infertility in modern medicine. The application of ART breaks through the conventional cognition that only natural reproduction can be performed in human history, and is a technical revolution in the field of reproductive medicine. Since the birth of the first example of test tube infants, ART is continuously updated, and has epoch-making significance for the development of life science. In recent years, sterility due to low sperm quality in men has been markedly increased, resulting in a great increase in ART demand. However, the ART has a low overall success rate at present, and the success rate is still improved by optimizing the conventional operation steps. The acquisition of high-quality sperms is not separated from the successful in-vitro fertilization once, and the optimization of the sperm quality before the combination with the ovum is the key for improving the ART success rate. In the ART process, after semen is obtained from male sterile patients, sperm is first optimized, then the optimized sperm is subjected to in vitro capacitation, and only sperm after successful capacitation can be combined with an ovum. Therefore, how to improve the in vitro capacitation efficiency of sperms becomes a key for improving the quality of sperms in the ART process, and is also a hot problem of research in the current assisted reproduction field.
Sperm culture compositions directly or indirectly affect the in vitro capacitation process of sperm, and currently, HTF is a sperm culture that is frequently used clinically. Therefore, the addition of new components to the HTF to promote the in vitro capacitation of the sperm and improve the sperm quality is a new idea for improving the ART success rate.
SERPINA5 (protein C inhibitor, PCI) is one of the serine protease inhibitor family members, is highly expressed in the male reproductive tract, and male serpin 5-deficient mice are sterile, suggesting that SERPINA5 plays an important role in spermatogenesis and male reproduction. The expression level of SERPINA5 in sperms of normal sperm parameters but sterile men is increased, and the SERPINA is positioned at the top of the acrosome of the ejected sperms or the sperms taken out from the epididymal tail, is combined with a precursor of the acrosome enzyme, inhibits the precursor from being released before the acrosome reaction, and avoids the damage of the structure of the sperms.
Disclosure of Invention
The invention aims to: the HTF liquid drop for improving the fertilization capacity of the mouse sperm and the preparation and application methods thereof are provided, and the in-vitro fertilization capacity of the mouse sperm can be effectively improved.
The technical scheme is as follows: the HTF liquid drop for improving the fertilization capacity of the mouse sperm contains 1-2 mug of mouse SERPINA5 synthetic protein in each 1mL HTF liquid drop.
As a further limiting scheme of HTF droplets, 1 μg of mouse SERPINA5 synthetic protein is contained in each 1mLHTF droplet.
The preparation method of HTF liquid drops for improving the fertilization capacity of mouse sperm comprises the following steps:
step 1.1, manufacturing HTF liquid drops with the volume of 200 mu L/drop in a culture dish, covering the HTF liquid drops with tissue paraffin oil, and then putting the HTF liquid drops into an incubator for balancing;
step 1.2, preparing the mouse SERPINA5 artificially synthesized protein powder into a mouse SERPINA5 artificially synthesized protein concentrate by using serum-free HTF, wherein the concentration of the mouse SERPINA5 artificially synthesized protein concentrate is 240-260 mug/mL;
and 1.3, adding the mouse SERPINA5 artificial synthetic protein into the HTF liquid drop to concentrate, so that the concentration of the mouse SERPINA5 artificial synthetic protein in the HTF liquid drop is 1-2 mug/mL.
As a further limiting scheme of the preparation method, in step 1.1, the environment of the incubator is controlled as follows: at a temperature of 37℃CO 2 The content is 5%.
As a further limiting scheme of the preparation method, in step 1.1, the time period of the equilibrium in the incubator is as follows: and 12-24 hours.
As a further limiting scheme of the preparation method, in the step 1.2, the concentration of the mouse SERPINA5 synthetic protein concentrate is 250 mug/mL.
As a further limiting scheme of the preparation method, in step 1.3, the concentration of the mouse SERPINA5 synthetic protein in the HTF droplet is 1 μg/mL.
The application method of the HTF liquid drop for improving the fertilization capacity of the mouse sperm comprises the following steps:
step 2.1, HTF droplets with the concentration of 1-2 mug/mL of mouse SERPINA5 synthetic protein are placed at 37 ℃ and CO 2 Preheating in an incubator with the content of 5%;
step 2.2, mixing the mouse sperms uniformly, adding the mixture into preheated HTF droplets, and placing the HTF droplets with the mouse sperms at 37 ℃ and CO 2 The energy is obtained in an incubator with the content of 5 percent for 0.5 to 1.5 hours.
As a further limiting scheme of the using method, in the step 2.1, the preheating time in the incubator is 0.5-1.5 hours.
As a further limiting version of the method of use, in step 2.2, the length of time of the energy obtained in the incubator is 1 hour.
Compared with the prior art, the invention has the beneficial effects that: according to the invention, the mouse SERPINA5 artificial synthetic protein is added into the mouse sperm capacitation liquid drop, so that the fertilization capacity of the mouse sperm after capacitation can be effectively improved; the invention has simple use method and can provide a reference method for optimizing sperm quality in the sperm in-vitro capacitation process of ART.
Drawings
FIG. 1 is a flow chart of a method of preparing HTF droplets of the present invention;
FIG. 2 is a graph showing the experimental result of fertilization rate of mouse sperm after mouse SERPINA5 artificial protein is added to HTF droplets;
FIG. 3 is a graph showing the experimental results of the ability of mouse sperm to bind to zona pellucida after the addition of mouse SERPINA5 artificial protein to HTF droplets of the invention.
Detailed Description
The technical scheme of the present invention will be described in detail with reference to the accompanying drawings, but the scope of the present invention is not limited to the embodiments.
Example 1:
the HTF liquid drop for improving the fertilization capacity of the mouse sperm contains 1-2 mug of mouse SERPINA5 synthetic protein in each 1mL HTF liquid drop.
As a further limiting embodiment of HTF droplets, preferably 1 μg of mouse SERPINA5 synthetic protein is contained per 1mL of HTF droplet.
As shown in fig. 1, the HTF droplet preparation method for improving fertilization ability of mouse sperm according to the present invention comprises the following steps:
step 1.1, manufacturing HTF liquid drops with the volume of 200 mu L/drop in a culture dish, covering the HTF liquid drops with tissue paraffin oil, and then putting the HTF liquid drops into an incubator for balancing;
step 1.2, preparing the mouse SERPINA5 artificially synthesized protein powder into a mouse SERPINA5 artificially synthesized protein concentrate by using serum-free HTF, wherein the concentration of the mouse SERPINA5 artificially synthesized protein concentrate is 240-260 mug/mL;
and 1.3, adding the mouse SERPINA5 artificial synthetic protein into the HTF liquid drop to concentrate, so that the concentration of the mouse SERPINA5 artificial synthetic protein in the HTF liquid drop is 1-2 mug/mL.
As a further limiting scheme of the preparation method, in step 1.1, the environment of the incubator is controlled as follows: at a temperature of 37℃CO 2 The content is 5%.
As a further limiting scheme of the preparation method, in step 1.1, the time period of the equilibrium in the incubator is as follows: and 12-24 hours.
As a further limiting scheme of the preparation method, preferably, in the step 1.2, the concentration of the mouse SERPINA5 synthetic protein concentrate is 250 mug/mL.
As a further limiting embodiment of the preparation method, preferably, in step 1.3, the concentration of the mouse SERPINA5 synthetic protein in the HTF droplet is 1. Mu.g/mL.
The application method of the HTF liquid drop for improving the fertilization capacity of the mouse sperm comprises the following steps:
step 2.1, HTF droplets with the concentration of 1-2 mug/mL of mouse SERPINA5 synthetic protein are placed at 37 ℃ and CO 2 Preheating in an incubator with the content of 5%;
step 2.2, mixing the mouse sperms uniformly, adding the mixture into preheated HTF droplets, and placing the HTF droplets with the mouse sperms at 37 ℃ and CO 2 The energy is obtained in an incubator with the content of 5 percent for 0.5 to 1.5 hours.
As a further limiting scheme of the using method, in the step 2.1, the preheating time in the incubator is 0.5-1.5 hours.
As a further limiting version of the method of use, in step 2.2, the length of time of the energy obtained in the incubator is 1 hour.
Experimental materials: ICR female mice of 8 months of age and ICR male mice of 12 weeks of age are clean grade and are approved by the ethical committee of animals. The mice are fed under specific conditions (22-28 ℃, relative humidity 50-70%, lighting for 12 hours a day, darkness for 12 hours) and are fed with the feed and drinking water at regular time.
And (3) experimental verification:
1. sperm acquisition and in vitro capacitation of male mice
(1.1) HTF (SAGE Co., USA) droplets, 200. Mu.L/drop, were made in petri dishes, and divided into seven groups, covered with tissue paraffin oil (SAGE Co., USA) and 5% CO at 37 ℃C 2 Is balanced in the incubator; HTF and M2 broth (Sigma Co., USA) were dispensed using 1.5mL Ep tubes at 37℃with 5% CO 2 Is balanced in the incubator of (a).
(1.2) adding mouse SERPINA5 artificial protein into HTF liquid drops every day, and the final concentration is as follows: 0 μg/mL (control), 1 μg/mL, 2 μg/mL, 3 μg/mL, 4 μg/mL, 5 μg/mL, and 10 μg/mL.
(1.3) killing 12 Zhou Lingxiong mice by cervical dislocation lethal method, spraying 75% alcohol on the abdomen, dissecting the lower abdomen, taking out epididymal tail, soaking in preheated HTF in (1), gently transferring with 1mL syringe under a split microscope to slowly swim out milky sperm mass, mixing all sperm uniformly and uniformly distributing into preheated HTF droplets in (1.1), placing HTF droplets with sperm at 37deg.C and 5% CO 2 The cells were allowed to harvest for 1 hour in an incubator.
2. Female mouse COCs acquisition and in vitro fertilization
(2.1) intraperitoneal injection of 10IU of pregnant horse serum gonadotropin (Pregnant Mare Serum Gonadotropin, PMSG, ningbo second hormone works) into each 8 month old ICR female rat, injection of 10IU of human chorionic gonadotropin (Human Choionic Gonadotophin, hCG, ningbo second hormone works) after 48 hours in the same manner, preparation of HTF droplet (100. Mu.L/droplet) and split oviduct culture solution (30. Mu.L/droplet) in a petri dish, respectively, of seven groups (1.2) each, tissue paraffin oil coverage, 5% CO at 37 ℃in a petri dish 2 Balancing in an incubator; m2 was dispensed with 1.5mL Ep tubes and equilibrated in a 5% CO2 incubator at 37 ℃.
(2.2) after hCG injection for 16 hours, the female mice were sacrificed by cervical dislocation lethal method, the abdomen was sprayed with 75% alcohol, the lower abdomen was dissected and the ampulla of the fallopian tube was immersed in M2, the enlarged part of the ampulla was gently allocated with a 1mL syringe under a split microscope, COCs clusters were drawn, and they were equally divided into preheated HTF droplets.
(2.3) seven groups of capacitation mice sperm of (1.3) were pipetted and added to corresponding HTF droplets to a final concentration of 5X 10 6 Drop at 37℃with 5% CO/mL 2 Incubate in incubator for 6 hours.
(2.4) aspiration of oocytes with oral pipettes, 3 washes in M2 and transfer into corresponding CM drops, 37℃C, 5% CO 2 Incubate in incubator for 18 hours.
(2.5) the two cell formation rates in each group were observed and counted under an inverted microscope.
3. Mouse sperm zona pellucida combination
(3.1) sperm acquisition of 12-week-old ICR Male mice and in vitro capacitation, and acquisition of 8-month-old ICR female COCs were identical (1.1) and (2.1), and were carried out at 37℃with 5% CO 2 1. Mu.g/mL hyaluronidase (Sigma Co., USA) was equilibrated in the incubator.
(3.2) the obtained 8 month old ICR female COCs were digested in 1. Mu.g/mL hyaluronidase (Sigma Co., USA) for 2 minutes, cumulus granulosa cells were removed, and the obtained oocytes were added to HTF droplets (10 oocytes per droplet) on average.
(3.3) seven groups of sperms after in vitro capacitation are sucked and respectively added into corresponding HTF liquid drops to make the final concentration of the sperms be 1 multiplied by 10 7 Drop at 37℃with 5% CO/mL 2 Incubate in incubator for 3 hours.
(3.4) the number of sperm bound to the zona pellucida of the oocyte was observed and counted under an inverted microscope.
4. Analysis of results
Experimental data were analyzed using SPSS19.0 and metering data were all used
The comparison between sample groups is shown by paired t test, P<0.05 indicates that the difference is statistically significant.
Compared with the in vitro fertilization rate of mice with no added protein group, after the in vitro fertilization of HTF liquid drops added with 1 mug/mL or 2 mug/mL of mouse SERPINA5 artificial protein is combined with the mouse COCs group, the in vitro fertilization rate of the mice oocytes is obviously increased (shown in figure 1, P < 0.05), wherein the effect of the 1 mug/mL group is obviously better than that of the 2 mug/mL group (shown in figure 1, P < 0.05). The result shows that the fertilization capacity of mouse sperms can be improved by adding a certain concentration of mouse SERPINA5 artificial protein into HTF liquid drops.
Compared with the mice without adding the protein group, the number of mouse sperms which are combined with the zona pellucida of each mouse oocyte after the HTF liquid drops added with 1 mug/mL or 2 mug/mL of mouse SERPINA5 artificial protein are capacitatively obtained in vitro is obviously increased (shown in figure 2, P < 0.05), wherein the effect of the 1 mug/mL group is obviously better than that of the 2 mug/mL group (shown in figure 2, P < 0.05). The result shows that the mouse SERPINA5 synthetic protein with a certain concentration is added into the HTF liquid drop to improve the binding capacity of mouse sperms and oocytes.
The in vitro fertilization rate is a direct index for reflecting the fertilization capacity of sperms, and the in vitro fertilization experimental result shows that compared with the in vitro fertilization experimental result without adding a proteome, the in vitro fertilization rate of oocytes after the combination of the sperms and the oocytes of the mice can be improved by adding a mouse SERPINA5 artificial protein with a certain concentration into HTF liquid drops, wherein the effect is optimal when the concentration of the mouse SERPINA5 artificial protein is 1 mug/mL. Sperm-egg binding capacity is a sensitive index of success or failure of fertilization, and is also a classical experiment reflecting the fertilization capacity of mouse sperm, and the result of the sperm-zona pellucida binding experiment shows that compared with the situation that a proteome is not added, the mouse SERPINA5 synthetic protein with a certain concentration is added in HTF liquid drops, so that the binding capacity of mouse sperm and oocyte can be improved, wherein the effect is optimal when the concentration of the mouse SERPINA5 synthetic protein is 1 mug/mL.
Therefore, the addition of mouse SERPINA5 synthetic protein with a certain concentration into HTF liquid drops can provide a reference method for optimizing sperm quality in the in vitro capacitation process of the sperm of ART by improving the in vitro fertilization capability of the mice sperm after capacitation.
As described above, although the present invention has been shown and described with reference to certain preferred embodiments, it is not to be construed as limiting the invention itself. Various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. An HTF droplet for improving fertilization capacity of mouse sperm is characterized in that each 1mL HTF droplet contains 1-2 mg mouse SERPINA5 synthetic protein.
2. The HTF droplet for enhancing fertilization capacity in a mouse sperm of claim 1, wherein each 1mL of HTF droplet comprises 1 mg mouse SERPINA5 synthetic protein.
3. The preparation method of the HTF liquid drop for improving the fertilization capacity of the mouse sperm is characterized by comprising the following steps:
step 1.1, manufacturing HTF liquid drops of 200 mL/drop in a culture dish, covering the HTF liquid drops with tissue paraffin oil, and then putting the HTF liquid drops into an incubator for balancing;
step 1.2, preparing the mouse SERPINA5 artificially synthesized protein powder into a mouse SERPINA5 artificially synthesized protein concentrate by using serum-free HTF, wherein the concentration of the mouse SERPINA5 artificially synthesized protein concentrate is 240-260 mug/mL;
and 1.3, adding the mouse SERPINA5 artificial synthetic protein into the HTF liquid drop to concentrate, so that the concentration of the mouse SERPINA5 artificial synthetic protein in the HTF liquid drop is 1-2 mg/mL.
4. The method for preparing HTF droplets for enhancing fertilization ability of mouse sperm according to claim 3, wherein in step 1.1, the environment of the incubator is controlled as follows: at a temperature of 37℃CO 2 The content is 5%.
5. The method for preparing HTF droplets for improving fertilization ability of mouse sperm according to claim 3, wherein in step 1.1, the time period for the equilibrium in the incubator is as follows: and (3) 12-24 hours.
6. The method for preparing HTF droplets for enhancing fertilization ability of mouse sperm according to claim 3, wherein in step 1.2, the concentration of the artificially synthesized protein in the mouse SERPINA5 is 250 μg/mL.
7. The method for preparing HTF droplets for enhancing fertilization ability of mouse sperm according to claim 3, wherein in step 1.3, the concentration of the mouse SERPINA5 synthetic protein in the HTF droplets is 1 mg/mL.
8. The application method of the HTF liquid drop for improving the fertilization capacity of the mouse sperm is characterized by comprising the following steps of:
step 2.1, placing HTF liquid drops with the concentration of 1-2 mg/mL of mouse SERPINA5 synthetic protein at 37 ℃ and CO 2 Preheating in an incubator with the content of 5%;
step 2.2, mixing the mouse sperms uniformly, adding the mixture into preheated HTF droplets, and placing the HTF droplets with the mouse sperms at 37 ℃ and CO 2 And obtaining energy in an incubator with the content of 5% for 0.5-1.5 hours.
9. The method for using HTF droplets to enhance fertilization of mouse sperm according to claim 8, wherein in step 2.1, the duration of preheating in the incubator is 0.5 to 1.5 hours.
10. The method of using HTF droplets to enhance fertilization capacity in a mouse sperm of claim 8, wherein the period of capacitation in the incubator in step 2.2 is 1 hour.
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