Experimental modeling of heterologous in vitro fertilization
Summary
Heterozygous in vitro fertilization experimental modeling can be used to: (1) simulate in vivo conditions of male and female gametes from different species of animals in mixed-table cultures to make their fusion and fertilization; (2) usually use the golden gopher eggs as the female gametes to experiment with a variety of mammalian or human sperm, so also known as sperm penetration assay (SPA).
Operation method
Sperm penetration test SPA
Principle
Heterologous in vitro fertilization (IVF) is a biotechnology technique in which male and female gametes from different species of animals are mixed and cultured to simulate in vivo conditions for fusion and fertilization. It is usually performed by using golden gopher eggs as female gametes and various mammalian or human sperms, so it is also called sperm penetration assay (SPA). The results of in vitro fertilization of human sperm with gopher eggs show a high positive correlation with in vitro fertilization of human homozygotes, and can be used to test the fertilizing ability of human sperm instead of human eggs.The SPA can reflect the functions of sperm capacitation, the acrosome reaction, and sperm-egg interactions, and therefore can be used to study the above aspects from the perspectives of physiology, pharmacology and toxicology.
Materials and Instruments
8～12 weeks old golden gopher Move 1 Experimental preparation and operation standard The whole experiment was prepared and operated according to the cell culture standards, and the steps involving eggs were carried out under the supervision of body-viewing microscope. 2 Animals 8-12 weeks old golden gopher; pure line feeding, supplemented with 20-30g/pc of fresh carrots per day; natural light, light/dark time ratio of about 14/10h; room temperature of 16-30℃. Rats of 8-20 weeks of age were selected for superovulation experiments. 3 Preparation of culture medium Generalized Bww culture medium was used. The preparation procedure was slightly modified from MarLinE. The reagents were all domestic analytical pure, triple-distilled water or distilled deionized water. The formula of storage solution is shown in Table 1, dissolve 1-4 in 800 ml of water; dissolve 5 in 50 ml of water alone and add it, and then dissolve 6-12 in turn, then the solution is red pH about 7.8. Store at 4℃ and use it within 2 months. Working solution (BWW) preparation procedure: l00ml of reserve solution dissolved in sodium bicarbonate 210mg, glucose l00mg, sodium lactate syrup (70-80) 0.325ml, human serum albumin 350mg. 1.0mol / LHEPES or sodium bicarbonate to adjust the pH 8.0 earth 0.1; 0.45μm and 0.22μm micropore filter membrane filtered successively; Packed in sealed packages and stored at 4℃, to be used within 1 week. 4 Gopher superovulation and egg preparation Female gophers were selected on day 1 of the estrous cycle (see vaginal milky white secretion), and were injected intraperitoneally with 30-40 IU of Pregnant Mare Serum Gonadotropin (PMSG) at 8-9 a.m. 56-61 h later with human chorionic villus (HCH). —61 h后注射人绒膜激素（hCG）等量；17h后断头解剖，取出输卵管放入预 冷 （4℃）BWW 中，针刺腹壶部取卵并洗净；0.1 %透明质酸酶消散颗粒细胞：洗净卵子 ；0.05% 胰酶消化透明带并洗净。 The resulting eggs were used immediately for insemination or placed in methylsilicone oil-covered Bww and stored at 4°C for 4 h for use. In one experiment, 23 gophers with similar conditions were divided into two groups and injected with hCG after 56 and 61 h of PMSG, respectively, and the number of ovulated eggs was counted after egg retrieval to compare the ovulation effects of the two groups. 5 Semen processing and spermatogenesis Human: Normal fertile young men were massaged to collect semen. Liquefied semen was filtered through double-layer teasing paper; 0.4 ml of semen was injected into the bottom of 2 ml of BWW (in a 5 ml test tube) and incubated at 37°C for 1 h. After standing up for 5 min, 80% of the upper layer of the liquid was sucked out, which was then obtained as the viable spermatozoa suspension. Centrifuge and wash for 2 times and adjust the sperm concentration to about l×l06/ml. Place lml of this suspension in a 1.5 ml plastic centrifuge tube and incubate in a sealed container (37°C, 5h) to enable spermatogenesis. Mice: 8-12 weeks old ICR mice, cut off the head, dissect out the epididymis, pick up the surrounding tissue, cut off the epididymis tail and put it into 0.2 ml of Bww and cut it into pieces; incubate at 37℃ for 5 min to make the sperms come out; take the sperms suspension and dilute it to (1-2)×l0ml and incubate it for 3h. 6 Insemination De-hyaline zoned gopher eggs were pre-cultured at 37℃ for 5min; 10-20 eggs were sucked and placed in a plastic petri dish (15mm×30mm,) to form a spherical droplet, injected with 50μL of energized spermatozoa, and then covered with a drop of sterilized methylsilicone oil to be incubated at 37℃ in saturated humidity for 2-3h. 7 Examination After insemination, the eggs were washed 3 times with Bww to remove the attached spermatozoa; the eggs were placed in the center of a 4-corner slide with a drop of special soft wax (petroleum jelly; paraffin I 9:l, V:V) and a marigold slide (2.1 mm × 2.1 mm). The 4 corners of the slide were pressed gently to flatten the egg; the egg was observed by phase contrast microscopy. Fertilization was determined by the presence of an enlarged translucent sperm head or two or more prokaryotic wells with sperm tails in the egg; Fertilization rate (FR) = number of fertilized eggs/number of eggs examined × 100. 8 Repeat experiments and quality control Repeated experiments were carried out according to the above procedures. There were 16 cases of human spermatozoa and 8 cases of mouse spermatozoa. For quality control, the same semen specimen was divided into 7 parallel samples in the same experiment, operated by different operators in parallel, and the FR was calculated after measurement, and the mean and coefficient of variation (CV) were calculated, and CV≤l5 was regarded as qualified]. Caveat The SPA experiment has many steps and is very demanding. The most critical steps are as follows： （1） All vessels should be strictly cleaned and sterilized according to cell culture requirements.(2) The process of egg collection and processing must be particularly precise and rapid. To avoid a decrease in egg activity, the oviducts can be placed in ice-cold (4 °C) BWW for several steps after removal and before enzymatic action; (3) Digestion with trypsin should be carried out in the presence of a small amount of water, and the eggs should be digested in the presence of a small amount of water.(3) Digestion with trypsin should be carried out under complete visual microscopic control, and the zona pellucida should be removed and cleaned when it is thin and loose, so as to avoid damage to the oocyte by trypsin； （4） The temperature should be controlled during sperm capacitation (37°C) and the sperm density should be adjusted to a suitable level (human: 10 X 10).10 X6Human: 10 X 106 /ml , Mouse: 1X10 X 10 6 /ml , mouse: 1 X 10 6 /ml6/ml), otherwise the fertilization rate will be affected； （5） When checking the fertilized eggs, the wax droplets at the four corners of the carrier sheet should be uniform in size, the softness and hardness should be adjusted appropriately with the room temperature, and the force at each corner should be consistent when pressing the sheet, so as to avoid excessive deformation or crumbling of the eggs, which may affect the observation. Common Problems Repeated experiments and quality control: Repeated experiments were carried out according to the above procedures. For quality control, the same semen specimen was divided into several parallel samples in the same experiment, and operated by different operators in parallel, and the FR was calculated after measurement, and the mean and coefficient of variation (CV) were calculated, and the CV ≤ 15% was considered to be qualified. For more product details, please visit Aladdin Scientific website.
Triple-distilled water Distilled deionized water Sodium chloride Potassium chloride Calcium chloride Streptomycin Sodium pyruvate Sodium hydroxide Sodium bicarbonate Sodium magnesium sulfate Dipotassium phosphate Phenol red Penicillin G Potassium G Glucose Sodium lactate syrup Human serum albumin Methyl silicone oil
Syringes Needles Scissors Test tubes Double teaser paper Centrifuge tubes Petri dishes Incubators Contrast microscope Coverslips Slides
The CV≤15% was considered as qualified. Source: "Establishment of experimental model of heterologous in vitro fertilization and its methodological discussion" Journal of Fujian Medical College.