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Apr 2021
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Gamete Fusion Assay in Mice
小鼠配子融合分析   

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Abstract

Gamete fusion, which is the final event of fertilization, is a crucial physiological event in the creation of a new fetus. In mammals, sperm IZUMO1 and oocyte IZUMO1R (JUNO) recognition play a role in triggering this process. Gamete fusion occurs through a complex but steady and unfailing intermolecular reaction because fertilization must ensure species specificity, in which fusion takes place between gametes of the same species only. Although many factors involved in this process have recently been identified, their specific contributions remain largely unknown. The current article describes detailed methods for assessment of gamete fusion in mice, visualized by fluorescent dye transfer, from unfertilized oocyte to spermatozoa. These methods are applicable not only for fixed cells but also live imaging of gametes.

Keywords: Spermatozoon (精子), Oocyte (卵母细胞), Fertilization (受精作用), Gamete fusion (配子融合), Mouse (小鼠)

Background

Mammalian fertilization is completed by cell-cell fusion, which is one of the most common physiological events. There are many obstacles that hinder the sperm’s capability to reach the oocyte. Among the enormous number of ejaculated spermatozoa (approximately 300 million in humans), only one spermatozoon is admitted to fertilize one oocyte.


Gamete recognition and fusion in mammals are part of a strict selection process in the molecular machinery of fertilization because gametes among different species are usually unable to merge with each other. These processes are likely carried out through a complex intermolecular interaction, in which izumo sperm-egg fusion 1 (IZUMO1) (Inoue et al., 2005), sperm acrosome associated 6 (SPACA6) (Lorenzetti et al., 2014; Barbaux et al., 2020; Noda et al., 2020), transmembrane protein 95 (TMEM95) (Lamas-Toranzo et al., 2020; Noda et al., 2020), fertilization influencing membrane protein (FIMP) (Fujihara et al., 2020), sperm-oocyte fusion required 1 (SOF1) (Noda et al., 2020), the newly-identified dendrocyte expressed seven transmembrane protein (DC-STAMP) domain-containing 1 (DCST1) and its paralogue DCST2 (Inoue et al., 2021) on the sperm side, and JUNO (also known as IZUMO1 receptor) (Bianchi et al., 2014) and cluster of differentiation 9 (CD9) (Kaji et al., 2000; Le Naour et al., 2000; Miyado et al., 2000) on the ovum side, all participate in gamete fusion, as proven by gene disruption. Among them, the IZUMO1-JUNO regulation system is likely essential for triggering gamete fusion (Inoue et al., 2013 and 2015; Aydin et al., 2016; Ohto et al., 2016), although how these factors contribute to gamete fusion remains unclear (Bianchi and Wright, 2020).


Mainly three different methods can be used for preparing zona pellucida (ZP)-free oocytes. The Acidic Tyrode's solution method is a general method that has been used by many researchers because preparation is quick and easy. However, long exposure to Acidic Tyrode’s solution causes the oocytes to die easily. Although in this study there were no obvious differences in the surface structure of Acidic Tyrode’s solution- and collagenase-treated ZP-free oocytes (Figure 2A), a remnant of dissolved ZP was found adhered on the oocyte plasma membrane only when the Acidic Tyrode’s solution method was used (Figure 2B), resulting in acrosome-intact spermatozoa binding to the surface of oocytes, which is considered a false binding (Yamagata et al., 2002). These problems are resolved by collagenase and mechanical ZP removal methods (Yamagata et al., 2002) (Figure 2B). However, regarding the mechanical ZP removal method, both expensive equipment and micromanipulator skills are required (Inoue and Okabe, 2008); therefore, the collagenase method is recommended as it is simple and does not require any specialized equipment or skill (Yamatoya et al., 2011).

Materials and Reagents

  1. 0.22-μm pore size polyethersulfone membrane (Merck, catalog number: S2GPU11RE)

  2. 1-ml, 26-gauge, 1/2-inch syringe (TERUMO CORPORATION, model: SS-01T2613S)

  3. 35-mm uncoated plastic dish plastic dish (IWAKI, model: 1000-035)

  4. 60-mm uncoated plastic dish plastic dish (IWAKI, model: 1010-060)

  5. ≥ 8-week-old female mice

  6. ≥ 12-week-old male mice

  7. Acidic Tyrode’s solution (Merck, catalog number: T1788)

  8. AlbuMAXTM I Lipid-Rich BSA (Thermo Fisher Scientific, catalog number: 11020021)

  9. CaCl2·2H2O (Merck, catalog number: C7902)

  10. Collagenase (FUJIFILM Wako Pure Chemical Corporation, catalog number: 038-22361)

  11. Equine chorionic gonadotropin (eCG) (ASKA Animal Health)

  12. FHM medium (Merck, catalog number: MR-024-D)

  13. Glucose (Merck, catalog number: G6152)

  14. Glutaraldehyde solution 20% (w/v) (FUJIFILM Wako Pure Chemical Corporation, catalog number: 072-02262)

  15. Hoechst 33342 (Thermo Fisher Scientific, catalog number: H1399)

  16. Human chorionic gonadotropin (hCG) (ASKA Animal Health)

  17. Hyaluronidase type IV-S (Merck, catalog number: H4272)

  18. KCl (Merck, catalog number: P5405)

  19. KH2PO4 (Merck, catalog number: P5655)

  20. MgSO4·7H2O (Merck, catalog number: 63138)

  21. Mineral oil (Merck, catalog number: M8410)

  22. NaCl (Merck, catalog number: S5886)

  23. NaHCO3 (Merck, catalog number: S5761)

  24. Penicillin-streptomycin (FUJIFILM Wako Pure Chemical Corporation, catalog number: 168-23191)

  25. Phenol red 0.04% (w/v) (FUJIFILM Wako Pure Chemical Corporation, catalog number: 163-20623)

  26. Sodium pyruvate (Merck, catalog number: P4562)

  27. Sterile water, endotoxin free (FUJIFILM Wako Pure Chemical Corporation, catalog number: 196-15645)

  28. TYH medium (see Recipes)

Equipment

  1. #5 forceps (DUMONT, model: 11252-20)

  2. Appropriate CO2 incubator (ASTEC, model: SCA-30DR) (for maintaining a 5% CO2 atmosphere)

  3. Appropriate fluorescent inverted microscope (e.g., Olympus, model: IX71)

  4. Appropriate stereo microscope (e.g., Olympus, model: SZX16)

  5. Egg-handling mouth pipette (DRUMMOND, model: 2-040-000), which has a joint with fire-polished glass tube of approximately 150-μm in diameter (DRUMMOND, model: 2-000-100) and a mouthpiece (MINATO MEDICAL CORPORATION, model: KA239-02), in accordance with instructions from a reputable textbook (e.g., Behringer et al., 2014) (Figure 1A)

  6. Fine scissors (Natsume Seisakusho, model: B12-H)

  7. Straight-blade Vannas scissors (Natsume Seisakusho, model: MB-54-1)

  8. Suitable thermal plate (e.g., TOKAI HIT, model: TPi-SZX2X) (it is advisable to use a thermal plate to maintain a temperature of 37°C on the stereo microscope in all experiments)

Software

  1. OriginPro 2020b (LightStone)

Procedure

  1. Preparation of oocytes

    1. Perform intraperitoneal injections of 7.5 IU eCG and 7.5 IU hCG at a 48-h interval to ≥ 8-week-old female mice of an appropriate strain, using a 1-ml, 26-gauge, 1/2-inch syringe [e.g., four hybrid B6D2F1 female mice for an experiment when wild-type mice are used (approximately 150 oocytes are ovulated)].

    2. Sacrifice the mice 15-17 h after hCG injection.

    3. Dissect the oviducts in accordance with instructions from a reputable textbook, using fine scissors and #5 forceps (e.g., Behringer et al., 2014).

    4. Transfer the oviducts to a mineral oil-covered 35-mm plastic dish.

    5. Newly ovulated oocytes, surrounded by cumulus cells [cumulus-oocyte complex (COC)], are found in the ampulla of the oviduct.

    6. Place one oviduct beside a mineral oil-covered 50-μl drop of TYH medium in a 60-mm plastic dish (Figure 1B and 1C).

    7. Use #5 forceps to grasp the oviduct, and supplementary forceps to create a tear in the oviduct close to where the oocytes are located (Figure 1C).



      Figure 1. Preparation prior to gamete fusion assay.

      A. Image of an egg-handling mouth pipette. B. Preparation of TYH drops. TYH medium is covered by pouring mineral oil after making TYH drops. C. Preparation of gametes. Oocyte preparation and spermatozoon preparation are shown in the top and bottom rows, respectively. These pictures correspond to Subheadings A and C, respectively. Scale bars: 1 mm.


  2. Removal of the zona pellucida (Figure 2)

    The gamete fusion assay is widely used for assessing the establishment of membrane fusion, for which most studies employ the Hoechst 33342 pre-load method. Please be careful during the ZP removal procedure prior to visualization of fused spermatozoa.

    1. Acidic Tyrode’s solution method

      1. Release the clutch of the COC into a 50-μl drop of TYH medium with 0.33 mg/ml hyaluronidase in a 60-mm plastic dish (Figure 1C).

      2. Allow a few COCs to incubate in a 50-μl drop of TYH medium with 0.33 mg/ml hyaluronidase at 37°C under 5% CO2 in air, until the cumulus cells are completely removed (within 5 min).

      3. Wash the cumulus-free oocytes by pipetting in and out using an egg-handling mouth pipette (Figure 1A), and subsequently transfer the oocytes into fresh 50-μl drops of TYH medium (repeat washing at least three times).

      4. Transfer the cumulus-free oocytes into a 50-μl drop of mineral oil-covered acidic Tyrode’s solution prepared in a 60-mm plastic dish.

      5. To remove the remnant medium, transfer the oocytes into a second 50-μl of acidic Tyrode’s solution.

      6. Repeatedly pipette the oocytes in and out until the ZP are dissolved under the stereoscopic microscope [it should occur within 30 s; otherwise, the survival rate of the oocytes will be significantly decreased (dead oocytes will be ruptured)].

      7. Wash the ZP-free oocytes at least three times by transferring them into fresh new 50-μl drops of TYH medium, to remove the remnant acidic Tyrode’s solution.

      8. Incubate the ZP-free oocytes in a 50-μl drop of TYH medium for more than 1 h at 37°C under 5% CO2 in air, to allow surface proteins to recover.

    2. Collagenase method

      1. Release the clutch of the COC into a 50-μl drop of TYH medium with 1 mg/ml of collagenase in a 60-mm plastic dish.

      2. Incubate for 30 min at 37°C under 5% CO2 in air.

      3. Carefully pick up the ZP-free oocytes with an egg-handling mouth pipette.

      4. Wash the ZP-free oocytes at least three times by transferring them into fresh new 50-μl drops of TYH medium.

      5. Maintain the ZP-free oocytes in TYH medium at 37°C under 5% CO2 in air until use.



      Figure 2. Comparison between acidic Tyrode’s solution and collagenase-treated ZP-free oocytes.

      A. High-resolution ultrastructure of the ZP-free oocytes imaged by a field emission scanning electron microscope (FE-SEM). There were no differences in the surface structure of oocytes obtained by the two methods. The yellow and red boxes correspond to each magnified image. Scale bars: 15 μm (top); 10 μm (middle); 5 μm (bottom). B. Visualization of a surface remnant of the dissolved ZP by monoclonal antibody (IE-3) against mouse ZP2, which is the major component of ZP. ZP-free oocytes prepared by the two methods were stained with 2 μg/ml IE-3. Subsequently, the antibody was detected using 5 μg/ml Alexa Fluor 647-labeled goat α-rat IgG (H+L) antibody. The images were taken with a confocal microscope. A remnant of the dissolved ZP was still found on the oocyte surface when the acidic Tyrode’s solution method was used, whereas they were undetectable when the collagenase method was employed. Scale bars: 100 μm.


  3. Collection of mature spermatozoa

    1. Dissect the cauda epididymis from ≥ 12-week-old male mice of an appropriate strain, using fine scissors and #5 forceps, in accordance with instructions from a reputable textbook (e.g., Behringer et al., 2014) (e.g., one hybrid B6D2F1 male mice for an experiment when wild-type mice are used).

    2. Transfer the cauda epididymis to a mineral oil-covered 35-mm plastic dish.

    3. Place the epididymis beside a 200-μl drop of TYH medium covered by mineral oil in a 60-mm plastic dish (Figure 1C).

    4. Use #5 forceps to grasp the cauda epididymis, and make a cut proximally to the thick ductus of the vas deferens, where motile spermatozoa are stored, with straight-blade Vannas scissors (Figure 1C).

    5. After squeezing the spermatozoa out from the cut end, hold a swarm of spermatozoa by sticking them to the tip of the supplementary forceps (Figure 1C).

    6. Introduce the swarm of spermatozoa into a 200-μl drop of TYH medium (Figure 1C).

    7. One hour after incubation, check sperm motility by observing the well-dispersed spermatozoa, and calculate the sperm concentration.

    8. Cultivate the spermatozoa for an additional 1 h in the TYH medium at 37°C under 5% CO2 in air, to induce capacitation and spontaneous acrosome reaction before insemination.


  4. Visualization of fused spermatozoa

    1. Prepare the ZP-free oocytes as described in Subheading B.

    2. Introduce the ZP-free oocytes into a 50-μl drop of 1 μg/ml Hoechst 33342 [the alternative reagent is a 4′,6-diamidino-2-phenylindole (DAPI) (Kaji et al., 2000)] TYH medium (up to 50 oocytes per spot) in a 60-mm plastic dish, and allow to stand for 10 min at 37°C under 5% CO2 in air.

    3. Transfer the oocytes into another fresh 50-μl drop of TYH medium covered with mineral oil.

    4. Incubate dye-loaded oocytes for 15 min at 37°C under 5% CO2 in air to release any excess dye.

    5. Repeat Steps 3 and 4 three more times, and subject the oocytes to the gamete fusion assay.

    6. Cultivate the spermatozoa as described in Subheading C to induce capacitation.

    7. Incubate dye-loaded oocytes with 2 × 105 spermatozoa/ml in a 100-μl drop of TYH medium for 30 min at 37°C under 5% CO2 in air.

    8. Transfer the sperm-bound eggs into a 50-μl drop of FHM medium containing 0.25% glutaraldehyde for slow fixation [fixation can be omitted at this stage for live imaging (Satouh et al., 2012)].

    9. Allow to stand for 5 min at room temperature.

    10. Wash the sperm-bound eggs by transferring them into fresh drops of FHM medium three times without pipetting in and out.

    11. Observe under a fluorescence microscope (20× or 40× objective lens, 405 nm excitation light). This procedure enables the staining of the nuclei of fused spermatozoa only, by transferring the dye into the spermatozoa after membrane fusion, whereas non-fused spermatozoa are undetectable. Chromosomes during meiosis metaphase II have the appearance of a cluster (Figure 3).

    12. Manually change several focuses of the inverted microscope, and count the number of fused spermatozoa per oocyte.



      Figure 3. Example of gamete fusion assay by the fluorescent dye transfer method: Dcst1/2 disrupted spermatozoa are unable to fuse with the oocytes.

      Fused spermatozoa were stained with Hoechst 33342 pre-loaded on the oocytes. The arrows and circles show the fused spermatozoa and meiosis metaphase II chromosomes, respectively. A few Dcst1/2+/- spermatozoa were successfully fused with the oocytes, whereas Dcst1/2-/- spermatozoa fusion with the oocytes never occurred. Scale bars: 100 μm.

Data analysis

Capture images with an appropriate fluorescent inverted microscope (e.g., Olympus IX71) and analyze them using the built-in software. After counting the fused spermatozoa under the microscope, perform statistical analyses using OriginPro 2020b (LightStone).

Recipes

  1. TYH medium

    6.976 g/L NaCl

    0.356 g/L KCl

    0.162 g/L KH2PO4

    1.0 g/L Glucose

    0.056 g/L Sodium pyruvate

    0.252 g/L CaCl2·2H2O

    0.294 g/L MgSO4·7H2O

    2.106 g/L NaHCO3

    4 g/L AlbuMAXTM I Lipid-Rich BSA

    10 ml/L Penicillin-streptomycin

    15 ml/L Phenol red 0.04% (w/v)

    Adjust to 1 L with sterile, endotoxin free water

    After filtering through a 0.22-μm pore size polyethersulfone membrane, dispense TYH medium to the appropriate volume of polystyrene conical tubes, and store at -20°C until use. Equilibrate TYH medium with CO2 before use.

Acknowledgments

The experimental procedures were based on a paper originally described by Inoue et al. (2021) (DOI: 10.7554/eLife.66313). This work was supported by JSPS KAKENHI Grant Number JP18H02453, and the joint research program of the Institute for Molecular and Cellular Regulation, Gunma University (19014).

Competing interests

The author declares no financial competing interests.

Ethics

All animal studies were approved by the Animal Care and Use Committee of Fukushima Medical University, Japan (approval number: 2020017) and performed under the guidelines and regulations of Fukushima Medical University.

References

  1. Aydin, H., Sultana, A., Li, S., Thavalingam, A. and Lee, J. E. (2016). Molecular architecture of the human sperm IZUMO1 and egg JUNO fertilization complex. Nature 534(7608): 562-565.
  2. Behringer, R., Gertsenstein, M., Nagy, K. V. and Nagy, A. (2014). Manipulating the Mouse Embryo: A Laboratory Manual. (4th Edition). Cold Spring Harbor Laboratory Press, NY.
  3. Bianchi, E., Doe, B., Goulding, D. and Wright, G. J. (2014). Juno is the egg Izumo receptor and is essential for mammalian fertilization. Nature 508(7497): 483-487.
  4. Bianchi, E. and Wright, G. J. (2020). Find and fuse: Unsolved mysteries in sperm-egg recognition. PLoS Biol 18(11): e3000953.
  5. Barbaux, S., Ialy-Radio, C., Chalbi, M., Dybal, E., Homps-Legrand, M., Do Cruzeiro, M. and Vaiman, D. (2020). Sperm SPACA6 protein is required for mammalian Sperm-Egg Adhesion/Fusion. Sci Rep 10(1): 5335.
  6. Fujihara, Y., Lu, Y., Noda, T., Oji, A., Larasati, T., Kojima-Kita, K., Yu, Z., Matzuk, R. M., Matzuk, M. M. and Ikawa, M. (2020). Spermatozoa lacking Fertilization Influencing Membrane Protein (FIMP) fail to fuse with oocytes in mice. Proc Natl Acad Sci U S A 117(17): 9393-9400.
  7. Inoue, N., Ikawa, M., Isotani, A. and Okabe, M. (2005). The immunoglobulin superfamily protein Izumo is required for sperm to fuse with eggs. Nature 434(7030): 234-238.
  8. Inoue, N. and Okabe, M. (2008). Sperm-egg fusion assay in mammals. Methods Mol Biol 475: 335-345.
  9. Inoue, N., Hamada, D., Kamikubo, H., Hirata, K., Kataoka, M., Yamamoto, M., Ikawa, M., Okabe, M. and Hagihara, Y. (2013). Molecular dissection of IZUMO1, a sperm protein essential for sperm-egg fusion. Development 140(15): 3221-3229.
  10. Inoue, N., Hagihara, Y., Wright, D., Suzuki, T. and Wada, I. (2015). Oocyte-triggered dimerization of sperm IZUMO1 promotes sperm-egg fusion in mice. Nat Commun 6: 8858.
  11. Inoue, N., Hagihara, Y. and Wada, I. (2021). Evolutionarily conserved sperm factors, DCST1 and DCST2, are required for gamete fusion. Elife 10: e66313.
  12. Kaji, K., Oda, S., Shikano, T., Ohnuki, T., Uematsu, Y., Sakagami, J., Tada, N., Miyazaki, S. and Kudo, A. (2000). The gamete fusion process is defective in eggs of Cd9-deficient mice. Nat Genet 24(3): 279-282.
  13. Lorenzetti, D., Poirier, C., Zhao, M., Overbeek, P. A., Harrison, W. and Bishop, C. E. (2014). A transgenic insertion on mouse chromosome 17 inactivates a novel immunoglobulin superfamily gene potentially involved in sperm-egg fusion. Mamm Genome 25(3-4): 141-148.
  14. Lamas-Toranzo, I., Hamze, J. G., Bianchi, E., Fernández-Fuertes, B., Pérez-Cerezales, S., Laguna-Barraza, R., Fernández-González, R., Lonergan, P., Gutiérrez-Adán, A. and Wright, G. J. (2020). TMEM95 is a sperm membrane protein essential for mammalian fertilization. Elife 9: e53913.
  15. Le Naour, F., Rubinstein, E., Jasmin, C., Prenant, M. and Boucheix, C. (2000). Severely reduced female fertility in CD9-deficient mice.Science 287(5451): 319-321.
  16. Miyado, K., Yamada, G., Yamada, S., Hasuwa, H., Nakamura, Y., Ryu, F., Suzuki, K., Kosai, K., Inoue, K., Ogura, A., Okabe, M. and Mekada, E. (2000). Requirement of CD9 on the egg plasma membrane for fertilization. Science 287(5451): 321-324.
  17. Noda, T., Lu, Y., Fujihara, Y., Oura, S., Koyano, T., Kobayashi, S., Matzuk, M. M. and Ikawa, M. (2020). Sperm proteins SOF1, TMEM95, and SPACA6 are required for sperm-oocyte fusion in mice. Proc Natl Acad Sci U S A 117(21): 11493-11502.
  18. Ohto, U., Ishida, H., Krayukhina, E., Uchiyama, S., Inoue, N. and Shimizu, T. (2016). Structure of IZUMO1-JUNO reveals sperm-oocyte recognition during mammalian fertilization. Nature 534(7608): 566-569.
  19. Satouh, Y., Inoue, N., Ikawa, M. and Okabe, M. (2012). Visualization of the moment of mouse sperm-egg fusion and dynamic localization of IZUMO1. J Cell Sci 125(Pt 21): 4985-4990.
  20. Yamagata, K., Nakanishi, T., Ikawa, M., Yamaguchi, R., Moss, S. B. and Okabe, M. (2002). Sperm from the calmegin-deficient mouse have normal abilities for binding and fusion to the egg plasma membrane. Dev Biol 250(2): 348-357.
  21. Yamatoya, K., Ito, C., Araki, M., Furuse, R. and Toshimori, K. (2011). One-step collagenase method for zona pellucida removal in unfertilized eggs: easy and gentle method for large-scale preparation. Reprod Med Biol 10(2): 97-103.

简介

[摘要]配子融合是受精的最终事件,是产生新胎儿的关键生理事件。在哺乳动物中,精子 IZUMO1 和卵母细胞 IZUMO1R (JUNO) 识别在触发该过程中起作用。配子融合是通过复杂但稳定且持续的分子间反应发生的,因为受精必须确保物种特异性,其中融合仅发生在同一物种的配子之间。尽管最近已经确定了参与该过程的许多因素,但它们的具体贡献在很大程度上仍然未知。当前文章描述了评估小鼠配子融合的详细方法,通过荧光染料转移可视化,从未受精的卵母细胞到精子。这些方法不仅适用于固定细胞,也适用于配子的实时成像。

[背景]哺乳动物受精是通过细胞-细胞融合,这是最常见的生理事件中的一个完成。有许多障碍会阻碍精子到达卵母细胞的能力。在数量庞大的射精精子中(人类大约有 3 亿个),只有一个精子可以使一个卵母细胞受精。

哺乳动物的配子识别和融合是受精分子机制中严格选择过程的一部分,因为不同物种之间的配子通常无法相互融合。这些过程可能通过复杂的分子间相互作用进行,其中 izumo 精子 - 卵子融合 1 (IZUMO1) (Inoue et al. , 2005)、精子顶体相关 6 (SPACA6) (Lorenzetti et al ., 2014; Barbaux et al. ., 2020; Noda et al ., 2020)、跨膜蛋白 95 (TMEM95) (Lamas-Toranzo et al. , 2020; Noda et al. , 2020)、受精影响膜蛋白 (FIMP) (Fujihara et al. , 20, 20) ),精卵融合需要 1 (SOF1)(Noda等人,2020 年),新鉴定的树突细胞表达了包含 7 个跨膜蛋白(DC-STAMP)结构域的 1(DCST1)及其旁系同源物 DCST2(Inoue等人,2020 年)。, 2021) 在精子方面,以及 JUNO(也称为 IZUMO1 受体)(Bianchi等人,2014 年)和分化簇 9(CD9)(Kaji等人,2000 年;Le Naour等人,2000 年;Miyado et al. , 2000) 在卵子方面,都参与了配子融合,正如基因破坏所证明的那样。其中,IZUMO1-JUNO 调节系统可能对触发配子融合至关重要(Inoue等人,2013 年和 2015 年;Aydin等人,2016 年;Ohto等人,2016 年),尽管这些因素如何促进配子融合仍然存在不清楚(Bianchi 和 Wright,2020 年)。

主要有三种不同的方法可用于制备无透明带 (ZP) 的卵母细胞。酸性 Tyrode 溶液法是许多研究人员使用的通用方法,因为制备快速简便。然而,长时间接触酸性 Tyrode 溶液会导致卵母细胞很容易死亡。尽管在这项研究中,酸性 Tyrode 溶液和胶原酶处理的无 ZP 卵母细胞的表面结构没有明显差异(图 2A),但只有当酸性 Tyrode 溶液时,才发现溶解的 ZP 残留在卵母细胞质膜上方法(图 2B),导致顶体完整的精子结合到卵母细胞的表面,这被认为是错误的结合(Yamagata等,2002)。这些问题通过胶原酶和机械 ZP 去除方法得到解决(Yamagata等,2002)(图 2B)。然而,关于机械 ZP 去除方法,需要昂贵的设备和显微操作技能(Inoue 和 Okabe,2008);因此,推荐使用胶原酶法,因为它简单且不需要任何专门的设备或技能(Yamatoya等,2011)。

关键字:精子, 卵母细胞, 受精作用, 配子融合, 小鼠

材料和试剂

1.     0.22-μm 孔径聚醚砜膜(默克,目录号:S2GPU11RE

2.     1 毫升、26 号、1/2 英寸注射器(TERUMO CORPORATION,型号:SS-01T2613S

3.     35 毫米无涂层塑料盘塑料盘(IWAKI,型号:1000-035

4.     60 毫米无涂层塑料盘塑料盘(IWAKI,型号:1010-060

5.     ≥ 8 周龄雌性小鼠

6.     ≥ 12 周龄雄性小鼠

7.     酸性 Tyrode 溶液(默克,目录号:T1788

8.     AlbuMAX TM I 富含脂质的 BSAThermo Fisher Scientific,目录号:11020021

9.     CaCl ·2H O(默克,目录号:C7902

10.  胶原酶(FUJIFILM Wako Pure Chemical Corporation,目录号:038-22361

11.  马绒毛膜促性腺激素 (eCG) (ASKA Animal Health)

12.  FHM 培养基(默克,目录号:MR-024-D

13.  葡萄糖(默克,目录号:G6152

14.  戊二醛溶液 20%w/v)(FUJIFILM Wako Pure Chemical Corporation,目录号:072-02262

15.  Hoechst 33342Thermo Fisher Scientific,目录号:H1399

16.  人绒毛膜促性腺激素 (hCG) (ASKA Animal Health)

17.  IV-S型透明质酸酶(默克,目录号:H4272

18.  KCl(默克,目录号:P5405

19.  KH PO (默克,目录号:P5655

20.  MgSO ·7H O(默克,目录号:63138

21.  矿物油(默克,目录号:M8410

22.  NaCl(默克,目录号:S5886

23.  NaHCO (默克,目录号:S5761

24.  青霉素-链霉素(FUJIFILM Wako Pure Chemical Corporation,目录号:168-23191

25.  酚红 0.04%w/v)(FUJIFILM Wako Pure Chemical Corporation,目录号:163-20623

26.  丙酮酸钠(默克,目录号:P4562

27.  无菌水,无内毒素(FUJIFILM Wako Pure Chemical Corporation,目录号:196-15645

28.  TYH 培养基(见食谱)

 

设备

 

1.     #5 镊子(DUMONT,型号:11252-20

2.     合适的 CO 2培养箱(ASTEC,型号:SCA-30DR)(用于维持 5% CO 2气氛)

3.     合适的荧光倒置显微镜(例如Olympus,型号:IX71

4.     合适的体视显微镜(例如Olympus,型号:SZX16

5.     取蛋口移液器(DRUMMOND,型号:2-040-000),其接头与直径约 150-μm 的火抛光玻璃管(DRUMMOND,型号:2-000-100)和吹嘴(MINATO MEDICAL CORPORATION,型号:KA239-02),根据知名教科书的说明(例如Behringer等人2014)(图 1A

6.     精细剪刀(Natsume Seisakusho,型号:B12-H

7.     直刃 Vannas 剪刀(Natsume Seisakusho,型号:MB-54-1

8.     合适的热板(例如TOKAI HIT,型号:TPi-SZX2X)(建议在所有实验中使用热板在体视显微镜上保持 37°C 的温度)

 

软件

 

1.     OriginPro 2020b (LightStone)

 

程序

 

A.    卵母细胞的制备

1.     使用1 毫升、26 号、1/2 英寸注射器每隔 48 小时对 8 周龄以上的适当品系雌性小鼠进行 7.5 IU eCG 7.5 IU hCG 的腹腔注射[例如,四只杂交 B6D2F1 雌性小鼠用于实验,当使用野生型小鼠时(大约 150 个卵母细胞排卵)]

2.     hCG 注射后 15-17 小时处死小鼠。

3.     根据信誉良好的教科书的说明,使用细剪刀和 #5 镊子(例如Behringer等人2014 年)解剖输卵管。

4.     将输卵管转移到覆盖矿物油的 35 毫米塑料盘中。

5.     新排卵的卵母细胞被卵丘细胞 [卵丘-卵母细胞复合体 (COC)] 包围,位于输卵管壶腹中。

6.     将一根输卵管放在 60 毫米塑料盘中的 50 微升 TYH 培养基中,放在一滴矿物油覆盖的旁边(图 1B 1C)。

7.     使用 #5 镊子抓住输卵管,并使用辅助镊子在靠近卵母细胞所在位置的输卵管中产生撕裂(图 1C)。

 

 

1. 配子融合试验前的准备工作。

A. 处理鸡蛋的嘴吸管的图像。B. TYH 滴剂的制备。TYH 培养基在制作 TYH 滴剂后浇上矿物油。C. 配子的制备。卵母细胞制备和精子制备分别显示在顶行和底行。这些图片分别对应于副标题 A C。比例尺:1 毫米。

B.    去除透明带(图 2

配子融合试验广泛用于评估膜融合的建立,大多数研究采用 Hoechst 33342 预加载方法。在融合精子可视化之前,在 ZP 去除过程中请小心。

1.     酸性泰罗德溶液法

a.     COC 的离合器释放到 50 微升 TYH 培养基中,在 60 毫米塑料盘中加入 0.33 毫克/毫升透明质酸酶(图 1C)。

b.     允许一些 COC 50 微升含有 0.33 毫克/毫升透明质酸酶的 TYH 培养基中在 37°C 下在 5% CO 2空气中孵育,直到完全去除卵丘细胞(在 5 分钟内)。

c.     使用卵子处理口移液器(图 1A)移入和移出来清洗无卵丘卵母细胞,然后将卵母细胞转移到新鲜的 50 微升 TYH 培养基中(重复洗涤至少 3 次)。

d.     将无卵丘的卵母细胞转移到在 60 毫米塑料盘中制备的 50 微升矿物油覆盖的酸性 Tyrode 溶液中。

e.     为了去除残留培养基,将卵母细胞转移到第二个 50 微升的酸性 Tyrode 溶液中。

f. 反复吸取卵母细胞,直到 ZP 在立体显微镜下溶解 [它应该在 30 秒内发生否则,卵母细胞的存活率会显着下降(死卵母细胞会破裂)]

g.     将无 ZP 的卵母细胞转移到新的 50 微升 TYH 培养基中,将其清洗至少 3 次,以去除残留的酸性 Tyrode 溶液。

h.     将不含 ZP 的卵母细胞在 50 微升 TYH 培养基中于 37°C 5% CO 2空气中孵育 1 小时以上,以恢复表面蛋白。

2.     胶原酶法

a.     COC 的离合器释放到 50 微升 TYH 培养基中,在 60 毫米塑料盘中加入 1 毫克/毫升胶原酶。

b.     在空气中5% CO 2下,在 37°C 下孵育 30 分钟。

c.     用处理鸡蛋的嘴吸管小心地拿起无 ZP 的卵母细胞。

d.     将无 ZP 的卵母细胞转移到新的 50 微升 TYH 培养基中,将其清洗至少 3 次。

e.     将无 ZP 的卵母细胞保持在 TYH 培养基中,温度为 37°C ,空气中含有 5% CO 2直至使用。

 

 

2. 酸性 Tyrode 溶液和胶原酶处理的无 ZP 卵母细胞之间的比较。

A. 场发射扫描电子显微镜 (FE-SEM) 成像的无 ZP 卵母细胞的高分辨率超微结构。两种方法获得的卵母细胞的表面结构没有差异。黄色和红色框对应于每个放大的图像。比例尺:15 μm(顶部);10微米(中);5 微米(底部)。B. 用抗小鼠 ZP2 的单克隆抗体 (IE-3) 观察溶解的 ZP 的表面残留物,ZP2 ZP 的主要成分。两种方法制备的无 ZP 卵母细胞用 2 μg/ml IE-3 染色。随后,使用 5 μg/ml Alexa Fluor 647 标记的山羊 α-大鼠 IgG (H+L) 抗体检测抗体。图像是用共聚焦显微镜拍摄的。当使用酸性 Tyrode's 溶液方法时,在卵母细胞表面仍然发现溶解的 ZP 的残余物,而当使用胶原酶方法时,它们检测不到。比例尺:100 μm

C.    成熟精子的收集

1.     从的≥12周龄雄性小鼠解剖附睾尾适当应变,用细剪刀和#5镊子,按照从有信誉的教科书的指令(例如,贝灵格等人2014 例如,一个混合B6D2F1 雄性小鼠,用于使用野生型小鼠的实验)

2.     将附睾尾转移到矿物油覆盖的 35 毫米塑料盘中。

3.     将附睾放在 60 毫米塑料盘中的 200 微升 TYH 培养基中,并用矿物油覆盖(图 1C)。

4.     使用 #5 镊子抓住附睾尾,并用直刀片 Vannas 剪刀在输精管的粗导管近端切开,在那里存储活动精子(图 1C)。

5.     从切割端挤出精子后,将一大群精子粘在辅助钳的尖端(图 1C)。

6.     将大量精子引入 200 μl TYH 培养基中(图 1C)。

7.     孵育1小时后,观察分散良好的精子,检查精子活力,计算精子浓度。

8.     在空气中5% CO 2下,在 37°C TYH 培养基中再培养精子 1 小时,以在受精前诱导获能和自发的顶体反应。

D.    融合精子的可视化

1.     按照副标题 B 中的描述制备无 ZP 的卵母细胞。

2.     将无 ZP 的卵母细胞引入 50 微升 1 微克/毫升 Hoechst 33342 [替代试剂是 4',6-二脒基-2-苯基吲哚 (DAPI) (Kaji等人, 2000)] TYH 培养基 (每个点最多 50 个卵母细胞)在一个 60 毫米的塑料盘中,并在 5% CO 2空气中在 37°C 下静置 10 分钟。

3.     将卵母细胞转移到另一滴用矿物油覆盖的新鲜 50 μl TYH 培养基中。

4.     在空气中5% CO 2下,在 37°C 下孵育染料加载的卵母细胞 15 分钟,以释放任何多余的染料。

5.     再重复第 3 步和第 4 步三次,并对卵母细胞进行配子融合试验。

6.     按副标题 C 所述培养精子以诱导获能。

7.     将染料负载的卵母细胞与 2 × 10 5精子/ml 100 微升 TYH 培养基中孵育30 分钟,在 37°C 下,在空气中的5% CO 2下。

8.     将精子结合的卵子转移到 50 微升含有 0.25% 戊二醛的 FHM 培养基中进行缓慢固定 [在此阶段可以省略固定以进行实时成像 (Satouh等人, 2012)] 

9.     在室温下静置 5 分钟。

10.  将受精卵转移到新鲜的 FHM 培养基中 3 次,无需移液进出。

11.  在荧光显微镜下观察(20× 40× 物镜,405 nm 激发光)。该程序仅通过在膜融合后将染料转移到精子中来对融合精子的细胞核进行染色,而无法检测到非融合精子。减数分裂中期 II 期间的染色体外观呈簇状(图 3)。 

12.  手动改变倒置显微镜的几个焦点,并计算每个卵母细胞融合精子的数量。

 

 

3. 荧光染料转移法配子融合分析示例:Dcst1/2破坏的精子无法与卵母细胞融合。

融合的精子用预装在卵母细胞上的 Hoechst 33342 染色。箭头和圆圈分别显示融合的精子和减数分裂中期 II 染色体。一些Dcst1/2 +/-精子成功地与卵母细胞融合,而Dcst1/2 -/-精子与卵母细胞的融合从未发生。比例尺:100 μm

 

 

数据分析秒

 

使用合适的荧光倒置显微镜(例如Olympus IX71)捕获图像并使用内置软件对其进行分析。在显微镜下对融合的精子进行计数后,使用OriginPro 2020b (LightStone)进行统计分析。

 

食谱

 

1.     TYH培养基

6.976 /升氯化钠

0.356 /升氯化钾

0.162 / KH PO 4

1.0 /升葡萄糖

0.056 g/L 丙酮酸钠

0.252 g/L CaCl ·2H O

0.294 g/L MgSO ·7H O

2.106 /升碳酸氢钠3

4 g/L AlbuMAX TM I 富含脂质的 BSA

10毫升/升青霉素-链霉素

15 ml/L 酚红 0.04% (w/v)

用无菌、无内毒素的水调节至 1 L

通过 0.22-μm 孔径聚醚砜膜过滤后,将 TYH 介质分配到适当体积的聚苯乙烯锥形管中,并储存在 -20°C 直至使用。使用前用CO 2平衡 TYH 培养基。

 

致谢

 

实验程序基于 Inoue等人最初描述的论文(2021) (DOI: 10.7554/eLife.66313)。这项工作得到了 JSPS KAKENHI 资助号 JP18H02453 和群马大学分子和细胞调节研究所的联合研究计划 (19014) 的支持。

 

利益争夺

 

作者声明没有经济上的竞争利益。

 

伦理

 

所有动物研究均经日本福岛医科大学动物护理和使用委员会批准(批准号:2020017),并在福岛医科大学的指导方针和规定下进行。

 

参考问题

 

1.     Aydin, H.Sultana, A.Li, S.Thavalingam, A. Lee, JE2016 年)。人类精子IZUMO1和卵子JUNO受精复合物的分子结构。 自然5347608):562-565

2.     Behringer, R.Gertsenstein, M.Nagy, KV Nagy, A.2014 年)。操纵小鼠胚胎:实验室手册。(第 4 版)。纽约冷泉港实验室出版社。

3.     Bianchi, E.Doe, B.Goulding, D. Wright, GJ2014 年)。Juno 是卵子 Izumo 受体,是哺乳动物受精所必需的。 自然508(7497)483-487             

4.     Bianchi, E. Wright, GJ2020 年)。发现并融合:精卵识别中未解之谜。 PLoS 生物学18(11)e3000953

5.     Barbaux, S.Ialy-Radio, C.Chalbi, M.Dybal, E.Homps-Legrand, M.Do Cruzeiro, M. Vaiman, D. (2020)精子 SPACA6 蛋白是哺乳动物精子-卵子粘附/融合所必需的科学报告10(1): 5335              

6.     Fujihara, Y., Lu, Y., Noda, T., Oji, A., Larasati, T., Kojima-Kita, K., Yu, Z., Matzuk, RM, Matzuk, MM Ikawa, M. ( 2020)。乏受精影响膜蛋白 (FIMP) 的精子无法与小鼠的卵母细胞融合。 Proc Natl Acad Sci USA 117(17): 9393-9400

7.     Inoue, N.Ikawa, M.Isotani, A. Okabe, M. (2005)精子与卵子融合需要免疫球蛋白超家族蛋白 Izumo自然4347030):234-238

8.     Inoue, N. Okabe, M. (2008)哺乳动物的精子-卵子融合试验。方法 Mol Biol 475335-345

9.     Inoue, N., Hamada, D., Kamikubo, H., Hirata, K., Kataoka, M., Yamamoto, M., Ikawa, M., Okabe, M. Hagihara, Y. (2013)IZUMO1 的分子解剖,IZUMO1 是精卵融合必不可少的精子蛋白。 发展140(15)3221-3229

10.  Inoue, N.Hagihara, Y.Wright, D.Suzuki, T. Wada, I.2015 年)。卵母细胞触发的精子 IZUMO1 二聚化促进小鼠精卵融合。国家通讯社68858

11.  Inoue, N.Hagihara, Y. Wada, I. (2021)配子融合需要进化上保守的精子因子 DCST1 DCST2Elife 10e66313

12.  Kaji, K., Oda, S., Shikano, T., Ohnuki, T., Uematsu, Y., Sakagami, J., Tada, N., Miyazaki, S. Kudo, A. (2000)Cd9 陷小鼠的卵子中的配子融合过程存在缺陷。 Nat Genet 243):279-282             

13.  Lorenzetti, D.Poirier, C.Zhao, M.Overbeek, PAHarrison, W. Bishop, CE2014 年)。小鼠 17 号染色体上的转基因插入使可能参与精卵融合的新型免疫球蛋白超家族基因失活。 哺乳动物基因组25(3-4)141-148

14.  Lamas-Toranzo, I., Hamze, JG, Bianchi, E., Fernández-Fuertes, B., Pérez-Cerezales, S., Laguna-Barraza, R., Fernández-González, R., Lonergan, P., Gutiérrez -Adán, A. Wright, GJ2020 年)。TMEM95 是哺乳动物受精所必需的精子膜蛋白。 Elife 9e53913

15.  Le Naour, F.Rubinstein, E.Jasmin, C.Prenant, M. Boucheix, C. (2000)严重降低 CD9 缺陷小鼠的雌性生育能力。 科学2875451):319-321

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17.  野田,T.,路,Y.,藤原,Y.,大浦,S.,小谷野,T.,小林,S.MatzukMM和伊川中,(2020)精子蛋白 SOF1TMEM95 SPACA6 是小鼠精卵融合所必需的。Proc Natl Acad Sci USA 117(21): 11493-11502              

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Copyright Inoue. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Inoue, N. (2021). Gamete Fusion Assay in Mice. Bio-protocol 11(22): e4233. DOI: 10.21769/BioProtoc.4233.
  2. Inoue, N., Hagihara, Y. and Wada, I. (2021). Evolutionarily conserved sperm factors, DCST1 and DCST2, are required for gamete fusion. Elife 10: e66313.
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