JPH0928721A - Method for promoting in-vitro fertilization effect of bovine spermatozoon - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method capable of promoting the in-vitro fertilization effect of the bovine spermatozoa. SOLUTION: This method comprises co-culturing the bovine spermatozoa with the bovine salpingian epitheliocytes, then culturing the same with the bovine ovum. The in-vitro fertilization power of the bovine spermatozoa is improved and, therefore, the productivity of the bovine in-vitro fertilized ovum is enhanced and the effective utilization of the frozen semen is made possible.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for promoting in vitro fertilization efficiency of bovine sperm. More specifically, the present invention relates to a method for promoting in vitro fertilization efficiency of bovine sperm, which is used when preparing an in vitro fertilized egg used in a fertilized egg transplantation method and the like.

[0002]

2. Description of the Related Art Conventionally, a fertilized egg (embryo) transplantation method has been used for the purpose of mass production of high-quality livestock and production of artificially fertilized eggs. As the fertilized egg transplantation method, the in-vivo fertilized egg transplantation method has been widely used. However, in the in-vivo fertilized egg transplantation method, it is difficult to stably supply the fertilized egg at low cost. Transplantation methods are being studied. That is, an in vitro fertilized egg transplantation method (generally, an egg is collected from a slaughtered female livestock, in vitro maturation, in vitro fertilization,
According to the method of transplanting to recipient livestock after the development of fertilized eggs), it is not necessary to perform operations such as introduction, breeding, and superovulation treatment of donor livestock, which is a high cost factor for in-vivo fertilized eggs. Fertilized eggs can be produced at a cost.
Further, in the case of in vitro fertilized egg production, eggs are collected from slaughtered livestock, and therefore there is an advantage that livestock eggs whose characteristics such as meat quality are known can be used. In vitro fertilization technology is essential for the production of in vitro fertilized eggs, and in the case of in vitro fertilization of cattle, frozen semen is generally used. Currently, as frozen semen used for in vitro fertilization, commercially available frozen semen is mainly used for artificial insemination. However, not all frozen semen collected from different breeds of cattle can be used for in vitro fertilization, and there are individual differences in in vitro fertilization ability among breeding cattle, and it has been reported that some semen can be used for in vitro fertilization and other semen is not. (Yoshiaki Minato, The 45th Annual Meeting of the Japanese Society of Veterinary Medicine, p53, 1992). Therefore, it has been attempted to improve and stabilize the fertilization rate by thawing frozen semen collected from different breeds of cow and using mixed semen obtained by mixing 3 to 5 frozen semen.

[0003]

As described above, since mixed semen is currently used for the production of bovine in-vitro fertilized fertilized eggs, it is difficult to produce fertilized eggs with a fixed lineage. That is, if semen collected from a superior bull can be used alone, a fertilized egg with a clear lineage can be obtained. For that purpose, it is necessary to obtain a high fertilization rate for any frozen semen, and a method of improving the fertilization efficiency of frozen semen is required. Studies on the improvement of the fertilization efficiency of bovine sperm have been widely carried out in the past.The outline is as follows: in order to fertilize a sperm with an egg, sperm motility and capacitat
ion) and the subsequent acrosome reaction are considered to be essential. So far, as a factor inducing the acquisition of fertility in vitro of bovine sperm (In vitro),
Calcium ionophore A23187 (Byrd, W., J. Exp. Z
ool., 215: 35-46, 1981), heparin (Parrish, JJ,
et al. Theriogenology, 25: 591-600, 1986), caffeine (Shioya, Y., et al. Theriogenology, 30: 489-
496, 1988) and bovine serum albumin (BSA) (Byrd, W., J. Ex.
p. Zool., 215: 35-46, 1981). At present, BO is used as a culture solution for bovine in vitro fertilization.
Liquid (Brackett, B G., & Oliphant, G., Biol. Reprod.,
12: 260-274, 1975) and Sp-TALP liquid (Parrish, JJ, e
Biol. Reprod., 38: 1171-1180, 1988) to which the above substances are added is generally used. Recently, PVA (polyv
It has been reported that bovine sperm can invade ova even in a protein-free medium containing no bovine serum albumin by adding inylalcohol) to the medium (Tajik, P., et al.
Biol. Reprod., 50: 1231-1237, 1994). Furthermore, they point out that the optimum concentration of sodium bicarbonate solution (46 mM) is important for increasing the rate of sperm invasion into eggs. Furthermore, when Minato et al. Mentioned above added theophylline to the medium for in vitro fertilization,
It has been reported that the fertilization of spermatozoa, which has a low in vitro fertilization rate, is improved as compared with the non-addition medium. As described above, various studies have been made on the preservation of bovine spermatozoa and the in vitro fertilization medium, but a method for further enhancing the fertilization efficiency of bovine spermatozoa is desired.

[0004]

In view of the above problems, the present inventors have established an in vitro fertilization system by improving the function of sperm and utilizing it effectively for in vitro fertilization. As a result of intensive studies on a method for efficiently producing eggs, the present invention has been completed by finding a method capable of promoting the fertilization efficiency of bovine sperm. That is, the method of the present invention comprises co-culturing bovine sperm with bovine oviductal epithelial cells, and then culturing with bovine oocytes for in vitro fertilization to promote in vitro fertilization efficiency of bovine sperm; The method for promoting in vitro fertilization efficiency of bovine spermatozoa as described above, which uses a thaw solution of semen.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, as bovine sperm, thawed bovine frozen semen is usually used, but semen collected from bovine may be used as it is.
The frozen semen may be thawed according to a conventional method. For example, the frozen semen stored in the straw may be thawed by immersing it in a warm bath at about 37 ° C. In addition, bovine oviductal epithelial cells can be obtained according to a conventional method. For example, bovine oviductal tissue is collected, the adipose tissue and connective tissue around it are removed, and washed with PBS ^- solution 2-3 times. Then, the oviduct is incised with scissors, and the epithelial cell layer on the inner surface of the oviduct is lightly scraped off with a scalpel. The fallopian tube epithelial cells thus obtained are preferably subcultured and stabilized.

Co-culture of bovine sperm and bovine oviductal epithelial cells
As long as the bovine sperm and the bovine oviductal epithelial cells are cultured in a state in which they can come into contact with each other, there is no particular limitation. RITC-807 containing fetal serum
Culture using a medium, etc.). After the epithelial cells are in a confluent state, a sperm suspension (sperm count: about 1 × 10 ⁶ ) is added to the well, and an appropriate medium (for example, RITC-807 medium containing fetal bovine serum) is added. Etc.) in 5% CO ₂ /95% air, 37 ° C., and saturated humidity atmosphere. The co-culture time is about 3 to 24 hours, preferably about 8 to 15 hours, more preferably about 12 hours. If the co-culture time is less than 3 hours, the effect of promoting fertilization efficiency is insufficient, and if it exceeds 24 hours, sperm motility may decrease, which is not preferable.

In vitro fertilization is carried out by culturing the spermatozoa thus co-cultured with bovine ova (mature ova) in an appropriate medium. The medium used is generally BO
Liquid (Brakett and Oliphant, Biol. Reprod., 12, 260-274, 19
75) is used. If necessary, heparin, caffeine, fatty acid-free bovine serum albumin, etc. may be added to the BO solution. The number of sperms at the time of in vitro fertilization can be appropriately adjusted according to the motility of sperms. Preferred fertilization conditions include 5 in a BO solution containing heparin, caffeine and fatty acid-free bovine serum albumin.
% CO ₂ /95% air, about 38.5 ° C., in a saturated humidity atmosphere, using a sperm count of about 5 × 10 ⁶ and culturing an egg for about 6 hours.

The bovine mature egg used in the above steps is
It is generally obtained by culturing an egg collected from bovine ovary for about 20 to 22 hours under the conditions of 5% CO ₂ /95% air and saturated humidity. The above-mentioned bovine egg collection is usually performed from the ovaries of slaughtered bovines. In vitro maturation of the egg is usually
The serum-free medium used for egg maturation performed in serum-free medium is TCM199 medium containing tumor growth factor-α (TGF-α, 1
0 ng / ml), insulin (5 μg / ml) and BSA (1 mg / ml)
A medium containing about 1 ml) is preferably used, but the medium is not limited to this and EG may be added to an appropriate basal medium.
A medium supplemented with growth factors such as F and TGF-α, insulin, BSA and the like can also be used.

[0009]

The present invention will be described in more detail based on the following examples, but the invention is not intended to be limited to these examples. Example 1 a) Bovine fallopian tube epithelial cells (BOEC), bovine endometrial cells (BUE)
C), bovine granulosa cells (BGC), fetal bovine pulmonary artery vascular endothelial cells
Cells (BFPA-EC) Isolation and culture of bovine oviduct epithelial cells (BOEC): calcium adult cattle oviduct collected by slaughterhouse, magnesium free phosphate buffered衡液(PBS ^-) of entered were placed in a centrifuge tube Cool to 4 ℃ and bring back to the laboratory.
Removes adipose and connective tissue around the fallopian tube tissue,
Wash 2-3 times with PBS ^- solution. Next, the oviduct is incised with scissors, and the epithelial cell layer on the inner surface of the oviduct is gently scraped off with a scalpel. The epithelial cells are suspended in a cell growth medium (DME / F12 + 10% fetal bovine serum), and centrifugation is repeated twice at 1,500 rpm for 5 minutes to wash the cells. A cell suspension is prepared by adding fresh cell growth medium to the cell pellet. In a 25 cm ² culture flask treated with collagen, the cell density is 0.5 ~ with 5 ml of medium.
Inject the cell suspension at 1 × 10 ⁶ cells / culture flask. The culture is performed in an incubator set to 5% CO ₂ /95% air, 37 ° C, and saturated humidity. After culturing for 5 to 7 days, the primary culture was performed when the cells proliferated and became confluent (the cells proliferated on the culture surface and became saturated in a monolayer). Subculture was carried out from the BOEC cells of the primary culture by the following method. First, after removing the medium, the cells are washed with PBS ^- solution. Trypsin-EDTA solution (PBS ^- solution to final concentration
Disperse the cells with 0.025% trypsin and 0.02% EDTA). A cell pellet is prepared by centrifugation at 1,500 rotations for 5 minutes, a medium for cell growth is added, and pipetting is performed to obtain a cell suspension of single cells. A collagen-treated 25 cm ² culture flask is prepared and subcultured by a 1: 2 to 1: 4 split method. In the experiment, cells at passage number 3 to 5 were used.

Bovine Endometrial Cells (BUEC): Adult bovine uteri collected at the slaughterhouse are brought back to the laboratory in the same manner as the fallopian tubes. Similar to the fallopian tube, the uterine cavity is opened with scissors, and the endometrial cells are scraped off with a scalpel. The method of culturing the endometrial cells and the medium are the same as those for the oviductal epithelial cells. In the experiment, cells at three passages were used.

Bovine granulosa cells (BGC): Adult ovaries collected at the slaughterhouse are brought back to the laboratory in the same manner as fallopian tubes. 3 ovaries
After washing with PBS ^- solution once, the follicles are incised with a scalpel and the eggs with granulosa cells attached are collected in a 90 mm Petri dish while observing under a microscope. Transfer the egg with granulosa cells attached to a centrifuge tube and pipette in PBS ^- solution to remove the granulosa cells from the egg. The cells are washed by repeating centrifugation at 1,500 rpm for 5 minutes twice, and then a cell growth medium consisting of DME / F12 + 10% fetal bovine serum is added to the cell pellet and stirred.
A part of the cell suspension is taken, the number of cells is counted with a hemocytometer, and 2 to 5 × 10 ⁵ cells are seeded in a 25 cm ² culture flask so that the amount of the medium is 5 ml. The primary culture is performed when the cells become confluent after 5 to 7 days of culture. Subculture is BOEC
Do the same as for cells. In the experiment, BGC cells at passage number 2 to 5 were used.

Fetal bovine pulmonary artery vascular endothelial cells (BFPA-EC):
Aseptically collect the pulmonary artery from a fetus of about 5 months using surgical scissors and tweezers, transfer to a clean bench, and then wash 3 times with PBS ^- solution containing heparin (10 μg / ml),
The blood remaining in the blood vessels was removed. The blood vessel was longitudinally incised with surgical scissors, spread on the rubber sheet with the inner surface facing up, and fixed with an injection needle. For drying prevention, sterile PBS ^- was dropped on the surface of the blood vessel spread, stripped-down endothelial layer lightly rubbing the intimal female surgical. And vascular endothelial cells attached to the female, the remaining vascular endothelial cell clumps on vessels PBS ^-
Cell pellets were collected by centrifugation at 1,500 rpm for 5 minutes. Make a cell suspension with RITC-807 (Far East Pharmaceutical Co., Ltd.) containing 5% fetal bovine serum and 100 ng / ml basic fibroblast growth factor (b-FGF), transfer to a 35 mm petri dish, and transfer to 5%.
CO _2/95% air, 37 ° C., were cultured for 5 days in a saturated humidity. After 5 days of culturing, when the cells became confluent, they were designated as primary cultures. Subculture is performed every 5 days, and the number of passages is 3 in the experiment.
˜5 times BPFA-EC cells were used. The isolated cells were positive for Von Willebrand antigen,
It was identified as a vascular endothelial cell by the intracellular uptake of Acetyl LDL.

B) Preparation of sperm The sperm used in Experiment 1 was frozen semen (A) collected from a bull (Japanese Black cattle). Remove the frozen semen from the liquid nitrogen tank and immediately add it to warm water at 37 ℃.
Soaked and melted for a minute. Thawed sperm (0.5 ml) contains Tyrode's sperm culture solution (Sp-TALP: Parrish, JJ et al. B).
iol. Reprod. 38, 1171-1180, 1988) 9 ml was added for dilution, and the mixture was washed by centrifugation at 2,000 rpm for 5 minutes. After washing the sperm again, a suspension of 10 ⁷ sperm / ml was prepared with 1 ml of Sp-TALP solution and co-cultured with BOEC, BUEC, BGC and BFPA-EC cells.

C) BOEC, BUEC, BGC, BFPA-EC cells co-cultured with bovine sperm Co-cultured BOEC, BUEC, BGC, BFPA-EC cells in about 24 Seed cells at 10 ⁴ cells / well and RITC-807 medium (Kyokuto Pharmaceutical Co., Ltd.)
The cells were cultured under the condition of 10% fetal bovine serum for 4 to 7 days. When each cell becomes confluent, b)
Add the sperm suspension prepared in, add 5% CO ₂ /95% air, 37
Co-culture was performed at ℃ and saturated humidity. About 1 x per well
Viability and motility of spermatozoa were measured after adding 10 ⁶ spermatozoa and coculturing in RITC-807 medium in a medium supplemented with 10% fetal bovine serum for 12 hours.

D) Measurement of sperm viability and motility The sperm co-cultured with BOEC, BUEC, BGC and BFPA-EC cells for 12 hours in c) were detached from each cell by pipetting and centrifuged at 2000 rpm for 5 minutes. The sperm were collected. At this time, the number of spermatozoa recovered in the suspension was 80 to 90% of the sperm sprouts.
%Met. The same amount of Sp-TALP containing 2% trypan blue was added to the collected sperm suspension, and the mixture was kept warm in a CO ₂ incubator at 37 ° C for 5 to 10 minutes, and then the sperm suspension was dropped on a slide glass, After enclosing with a cover glass, it was observed with a Nomarski differential interference microscope. The viability of spermatozoa was determined by the presence or absence of trypan blue staining, to determine live cells and dead cells, and expressed as the ratio of live cells to the total number of sperms. The sperm motility was represented by the ratio of “motile sperm” to the total number of spermatozoa, with all the spermatozoa that were flagellating as “motile sperm”. The viability and motility of sperm after freeze-thawing were measured immediately without culturing the prepared sperm suspension.

Experimental Results 1 The viability and motility of sperm (A) when co-cultured with different somatic cells were examined. Table 1 shows the results. Surviving sperm immediately after freeze-thawing was 68.2%, and moving sperm was 54.1%.
%Met. The survival rate of spermatozoa was 6.5% when the spermatozoa were cultured only in the medium for 12 hours without co-culture, whereas the survival rate of the spermatozoa when co-cultured with each somatic cell was BOEC, BUE.
47.5%, 22.6%, 1 for C, BGC, and BFPA-EC, respectively
It was 2.7% and 9.7%. In addition, the proportion of motility sperm was 39.8%, 15.3%, 7.4, compared to 2.5% in the medium alone.
% And 6.0%. It was found that sperm viability and motility were significantly improved by co-culture with somatic cells.
Among them, the spermatozoa co-cultured with BOEC had the highest survival and motility values. These results suggest that the effect of maintaining viability and motility of sperm, which is considered to be an important sperm function for fertilization, is specific to fallopian tube epithelial cells (BOEC).

[0017]

[Table 1]

Example 2In vitro fertilization capacity of individual sperm co-cultured with BOEC  BOEC is the most effective cell for sperm viability and motility
It was found that in vitro fertilization ability was
By co-culturing BOEC and sperm in low frozen semen
We examined whether external fertility could be improved. a) Collection of bovine follicular ova After slaughter, the collected adult cow ovaries are placed in a plastic bag.
Put it in, warm it with warm water at 30-35 ℃, and within 1-2 hours
It was carried to the laboratory and used for the experiment. Kept warm at 38.5 ° C
Sterile calcium- and magnesium-free phosphate buffer solution (PBS
^-), Wash the ovary well, then divide the ovary in two with ophthalmic scissors.
did. The ovaries were cultured in TCM199 medium with 25 mM HEPES, heparin (15
μg / ml), sodium pyruvate (1.25 mM), and antibiotics
Contains medium containing gentamicin (10 μg / ml) as quality
It was transferred into a 90 mm petri dish. Surgical meme with 3 pieces
It was cut into small pieces and the follicles were opened. Egg-containing liquid mesh
With multiple layers of cumulus / granulosa cells after filtration with
Was collected and used for the experiment. Cumulus / Granulosa cell-attached egg
After washing several times with TCM199 medium, about 30 drops per drop
Each was transferred to a maturation medium. For in vitro maturation culture, 60
Place each medium covered with mineral oil on the mm dish.
The amount of medium was 350 μl per drop. In serum-free medium
For in vitro maturation by TGF-α (10 ng / ml),
Add insulin (5 μg / ml) and BSA (1 mg / ml)
Using. BSA is TGF-α contained in the prepared liquid medium.
And insulin are prevented from being adsorbed in the storage container.
Added to In vitro maturation is 5% CO_Two/ 95% air, satiety
The culture was performed for 22 to 24 hours under the condition of the humidity.

B) In vitro fertilization In addition to the frozen semen (A) used in Example 1, two types (B,
In vitro fertility was examined individually for 3 spermatozoa in combination with C). Positive control (0 hour fresh medium)
As a procedure, thaw frozen semen (0.5 ml straw) of each Japanese Black Cattle in warm water at 37 ℃, add 6 ml of BO solution containing heparin (15 μg / ml) and caffeine (5 mM), and centrifuge twice. The sperm were washed repeatedly. The sperm was resuspended in a BO solution containing heparin and caffeine, and the sperm was measured with a hemocytometer to adjust the sperm concentration to 1 × 10 ⁷ / ml. This sperm fluid 50μ
l is caffeine (5 mM), fatty acid-free bovine serum albumin
It was added to 50 μl of BO solution containing (BSA, 10 mg / ml). Eggs that have undergone maturation culture contain caffeine (5 mM) and BSA (10 mg / ml) B
After washing 3 times with the O liquid, it was added to the sperm suspension. 5% at 38.5 ° C
Fertilization was performed by culturing for 6 hours under the conditions of CO ₂ /95% air and saturated humidity. The number of sperm in the culture at the time of fertilization is approximately 5 x
10 ⁶ cells / ml, caffeine (5 mM), heparin (7.5 μg / ml), BS
The condition is A (5 mg / ml). On the other hand, as an experimental group that had been cultured for 12 hours, one that was co-cultured with BOEC cells and "the fresh medium" that was not co-cultured as a control were examined.
The co-culture conditions were as in Experiment 1 c). After culturing for 12 hours, sperm were collected by pipetting, 2000 rpm,
The sperm was collected by centrifugation for 5 minutes. In the subsequent operation, in vitro fertilization was performed in the same manner as in the 0-hour fresh medium.

C) In vitro developmental culture After in vitro fertilization, 30 eggs were cultured in 350 μl drops containing the serum-free medium used for in vitro maturation. The 60 mm culture dish was pretreated with 150 μg / ml type I collagen. Culture conditions: 38.5 ℃, 5%
CO _2/95% air and saturated humidity. After culturing for 44 to 48 hours, the developmental status of 2-cell stage embryos was examined as an index of cleavage rate.

Experimental Result 2 In vitro fertilization was immediately performed on three kinds of semen freeze-thawed by a usual method to examine the cleavage rate (see Table 2). Semen A, B,
The cleavage rate for C is 58.9%, 20.2%, 12.2%,
Clearly, there was a large difference in in vitro fertilization among individuals. When the spermatozoa were cultured for 12 hours in vitro only before in vitro fertilization, the semen ratios of semen A, B and C were 2.0% and 0.0
%, 0.0%, which is extremely low. It is considered that this is due to the fact that the viability and motility were remarkably reduced by culturing the sperm only in the medium for 12 hours as shown in Table 1. However, when sperm were co-cultured with oviductal epithelial cells, the semen cleavage rates of semen A, B, C were 66%, 63.1%, 36.4%.
%, And a high value was obtained. With respect to spermatozoa (B, C) having inferior fertilizing ability by the usual in vitro fertilization method, a particularly remarkable effect of improving in vitro fertilizing ability was observed. By using this co-culture system of oviductal epithelial cells and spermatozoa, it is considered possible to effectively use frozen semen that could not be used for in vitro fertilization.

[0022]

[Table 2]

[0023]

As described above, according to the present invention, the in vitro fertilization ability of bovine sperm can be improved, so that the productivity of bovine in vitro fertilized eggs can be remarkably enhanced. Especially,
Frozen semen, which has a low fertility and cannot be used for in vitro fertilization, can be used, so that frozen semen can be effectively used. Furthermore, unlike the conventional method, it is not necessary to mix and use frozen semen collected from different breeding cows, and semen collected from breeding cows with a clear pedigree can be used alone, so that a fertilized egg with a fixed pedigree can be obtained. It has the advantage of being able to.

Claims (2)

[Claims] 1. A method for promoting in vitro fertilization efficiency of bovine sperm, which comprises co-culturing bovine sperm with bovine oviductal epithelial cells and then culturing with bovine oocytes for in vitro fertilization. 2. The method for promoting in vitro fertilization efficiency of bovine sperm according to claim 1, wherein a thawed solution of bovine frozen semen is used as bovine sperm.