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Ex vivo Follicle Rupture and in situ Zymography in Drosophila
果蝇离体卵泡破裂和原位酶谱   

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eLIFE
Dec 2017

Abstract

Ovulation, the process of releasing a mature oocyte from the ovary, is crucial for animal reproduction. In order for the process of ovulation to occur, a follicle must be fully matured and signaled to rupture from the ovary. During follicle rupture in both mammals and Drosophila, somatic follicle cells are enzymatically degraded to allow the oocyte to be liberated from the follicle. Here, we describe a detailed protocol of our newly developed ex vivo follicle rupture assay in Drosophila, which represents a first assay allowing direct quantification of follicles’ capacity to respond to ovulation stimuli and rupture. This assay can be modified to stimulate rupture with other reagents (for example, ionomycin) or to query enzymatic activity (in situ zymography). In addition, this assay allows genetic or pharmacological screens to identify genes or small molecules regulating follicle rupture in Drosophila.

Keywords: Drosophila (果蝇), Ovulation (排卵), Follicle rupture (卵泡破裂), Octopamine (章鱼胺), in situ zymography (原位酶谱法), Follicle cells (卵泡细胞)

Background

The study of ovulation in Drosophila has largely been limited due to technical challenges in direct visualization and quantification of ovulation events. In the last several decades, multiple indirect methods have been developed with limitations. The first method for the study of ovulation in Drosophila is to push the female’s abdomen and record if an egg was ejected from the ovipositor (Aigaki et al., 1991; Lee et al., 2003). This method is a rough estimate of whether there’s an ovulated egg inside the uterus. The second common assay is to measure the egg-laying capacity of female flies after mating. The egg-laying process is complex, involving development and maturation of a follicle, ovulation, transportation of the ovulated egg through the oviduct, selection of an appropriate egg-laying substrate, and oviposition (Spradling, 1993; Bloch Qazi et al., 2003). If any of these processes are impaired, it will result in defective egg laying. Another indicator that ovulation is impaired is an egg-retention phenotype (Monastirioti et al., 1996; Monastirioti, 2003; Cole et al., 2005). If a female has an excess of mature follicles within her ovaries, it indicates an anovulatory phenotype. However, this can be caused directly by an ovulation defect or indirectly by a defect downstream of ovulation in the egg-laying process. On the other hand, a lack of egg-retention phenotype does not necessarily mean a lack of ovulation defect. A fourth type of assay used to study ovulation is to examine if an egg is present in the reproductive tract (Heifetz et al., 2000; Lee et al., 2009; Lim et al., 2014). Variations in this assay range from quantifying ovulation rate by the percentage of females with an egg in their lower oviduct/common oviduct/uterus post mating (Lee et al., 2009; Lim et al., 2014), to examining the distribution of eggs in each of these separate regions over time (Heifetz et al., 2000). However, each of these assays could be influenced by the speed of oogenesis, ovulation, and oviposition. To account for all the possible drawbacks of each individual assay, we recently combined the egg-retention assay, the egg-laying assay, and the egg location in the female reproductive tract to estimate the average time for ovulating an egg (ovulation time; Sun and Spradling, 2013; Deady et al., 2015 and 2017; Deady and Sun, 2015; Knapp and Sun, 2017). However, this method is tedious and also relies on the indirect measurements of ovulation.

We recently characterized ovulation at a cellular level and discovered that Drosophila ovulation involves a follicle rupture process. During ovulation, posterior follicle cells activate matrix metalloproteinase 2 (Mmp2), which degrades posterior follicle cells allowing for the encased oocyte to rupture into the oviduct (Deady et al., 2015). We also found that this process is initiated by direct octopamine (OA) and octopamine receptor in mushroom body (Oamb) signaling in follicle cells, and the entire process can be recapitulated in our ex vivo culture system (Deady and Sun, 2015). We named this assay ex vivo follicle rupture, in which mature follicles are isolated from the ovary and stimulated with OA to induce follicle rupture. Percent of follicles ruptured can be reported at the end of a short three-hour incubation, which is a direct quantification of follicle rupture. This assay allows for a relatively simple, high-throughput examination of follicle rupture in Drosophila, and is ideal for genetic and pharmacological screens. However, this experiment is done ex vivo, and results should be verified in vivo using some of the assays described above.

Materials and Reagents

  1. Utility boxes, 500 ml, Nalgene (Thermo Fisher ScientificTM, catalog number: 5700-0500 )
  2. Aluminum foil
  3. Paper towel
  4. PYREXTM spot plates (9-well) (Corning, PYREX®, catalog number: 7220-85 )
  5. Stainless steel needles, 0.25 mm, 36 mm (Ted Pella, catalog number: 13561 )
  6. Plastic transfer pipets, disposable, 5.8 ml (Fisher Scientific, Fisherbrand, catalog number: 13-711-9CM )
  7. 1.5 ml Eppendorf tubes
  8. Grace’s Media, with L-Glutamine (Genesee Scientific, catalog number: 25-516G )
  9. Dry yeast, active (Genesee Scientific, catalog number: 62-103 )
  10. Cornmeal (Genesee Scientific, catalog number: 62-101 )
  11. Molasses (Fisher Scientific, catalog number: NC9109740)
    Manufacturer: LabScientific, catalog number: FLY-8008-16 .
  12. Agar (Genesee Scientific, catalog number: 66-103 )
  13. Tegosept (Genesee Scientific, catalog number: 20-258 )
  14. Ethanol
  15. Propionic acid (Sigma-Aldrich, catalog number: P1386-1L )
  16. Fetal bovine serum (Atlanta Biologicals, catalog number: S11150 )
  17. Penicillin-streptomycin (10,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  18. Octopamine hydrochloride (Sigma-Aldrich, catalog number: O0250-1G )
  19. Fluorescein-conjugated DQTM Gelatin From Pig Skin (Thermo Fisher Scientific, InvitrogenTM, catalog number: D12054 )
  20. Wet yeast paste (see Recipes)
  21. Fly food (see Recipes)
  22. Culture media mixture (see Recipes)
  23. Octopamine stock solution (see Recipes)
  24. Octopamine working solution (see Recipes)
  25. Fluorescein-conjugated DQTM Gelatin stock solution (see Recipes)
  26. Gelatin working solution (see Recipes)

Equipment

  1. Dumont #5 forceps (Fine Science Tools, catalog number: 11252-20 )
  2. Pipetman kit G series (P20, P200, P1000) (Gilson, catalog number: F167900 )
  3. Modular stereo microscope for fluorescent imaging (Leica Microsystems, model: Leica MZ10 F )
  4. sCMOS camera (PCO. 4.2)
  5. 29 °C incubator with humidity control–set to ~70% RH (Percival Scientific, model: DR-36VL )
  6. CO2 tank, CD 50 (Airgas, model: CGA-320 )
  7. Autoclave
  8. Flypad (Genesee Scientific, catalog number: 59-114 )
  9. Nutator (Fisher Scientific, model: 260100F )

Software

  1. ImageJ (NIH) (Schneider et al., 2012)
  2. Micro-Manager (NIH) (Schneider et al., 2012; Edelstein et al., 2014)

Procedure

  1. Fly rearing
    1. Genetic requirements
      A fluorescent reporter expressed in mature follicle cells (stage 14) is required for isolating mature follicles with an intact follicle-cell layer. In our experiments, we used either R47A04-Gal4 or R44E10-Gal4 from the Janelia Gal4 collection (Pfeiffer et al., 2008) to drive the expression of a UAS-RFP reporter specifically in stage-14 follicle cells.
    2. Collect virgin females with correct genotype and age them for 5-6D at 29 °C.
    3. Supplement food with ~1 teaspoon of fresh yeast paste 2-3D before the experiment.

  2. Experimental preparation
    1. Warm Grace’s media to room temperature (~25 °C).
    2. Prepare the culture media mixture (see Recipes).
    3. Line utility boxes with aluminum foil to shield inside from the light.
    4. Add a damp paper towel to the bottom of the box to maintain moisture during the three-hour incubation.

  3. Dissection and isolation (see Figure 1)
    1. Make sure to minimize the amount of time from the start of dissection to the last step in Dissection and Isolation. Limit the entire Dissection and Isolation within one hour.
    2. Fill one well in the PYREX spot plate with ~1 ml Grace’s media (Figure 1, ‘1. Initial dissection’).


      Figure 1. PYREX 9-well spot plate setup

    3. Use forceps to dissect ovary pairs out of eight females in Grace’s media. Make sure not to damage ovaries and keep them intact.
      Tip: Gently remove ovaries from the abdomen by grabbing the oviduct/uterus region instead of directly grabbing the ovaries.
    4. Fill another well with ~1 ml Grace’s media and transfer all ovary pairs into this new well (Figure 1, ‘2. Holding ovary pairs’).
    5. See Video 1. ‘Dissection Example’
      1. Move ovary pairs (~two at a time) to a new well with ~1 ml Grace’s media (Figure 1, ‘3. Isolating follicles’).
      2. Use one of the forceps to hold the anterior end of the ovary, near the germarium. Use the other forceps to gently separate ovarioles at the posterior end of the ovary near the calyx.
      3. Next, use the other forcep to gently squeeze follicles out of the posterior end. This process is to liberate follicles from surrounding ovariole sheath. Be careful not to puncture follicles.

      Video 1. Dissection example

    6. Identify mature follicles according to the fluorescent reporter and make a pile of intact mature follicles using the needle.
    7. Transfer the intact mature follicles using the P20 pipette to a new well filled with ~1 ml culture media (Figure 1, ‘4. Pool of isolated, intact S14 follicles’). Coat the pipet tip with culture media first to prevent follicles from sticking to the pipet tip.
    8. Repeat Steps C4-C7 until you have enough follicles or until around ~40 min have passed since initial dissection.
    9. Remove any younger-stage follicles in the pool and double check that every follicle in the pool remains intact. Younger-stage follicles should not express the fluorescent reporter when viewed under fluorescent light but can be viewed under the bright-field light.
    10. Use the needle to pile ~25-35 intact follicles together. Transfer piled follicles in culture medium to new wells with a P20 pipette. These follicles should not be dried–they should remain in the ~20 μl culture media in which they were transferred. Repeat this step until all of selected follicles are dispensed in new wells in ‘groups’ of 25-35, or until a total of ~50 min have passed since initial dissection (Figure 1, ‘~25-35 intact S14 follicles in ~20 μl CM’).
    11. Prepare octopamine working solution (see Recipes)–1 ml per each ‘group’.
    12. Add 1 ml of octopamine working solution to each well to stimulate ex vivo rupture OR 1 ml of culture media to each well as a negative control.
    13. Put the PYREX spot plate into a foil-lined utility box (prepared in Procedure B), cover the box, and put the box into a 29 °C incubator for three hours.

  4. Image acquisition
    1. Remove the box from the incubator after three hours.
    2. Use the needle to pile follicles toward the center of each well under a microscope. Do not overlap follicles with each other.
    3. Use Micro-Manager to capture both bright-field and fluorescent images.

  5. Modify for in situ zymography
    1. Follow Steps C1-C10 to prepare isolated mature follicles.
    2. Prepare gelatin working solution (see Recipes)–1 ml per each ‘group’.
    3. Add 1 ml of gelatin working solution to each well to stimulate ex vivo rupture and detect gelatinase activity. A negative control should be gelatin working solution without octopamine.
    4. After three hours incubation at 29 °C, wash out gelatin working solution from each well by pipetting out the solution and replacing with fresh culture media (be careful not to remove follicles from each well).
    5. Use the needle to pile follicles toward the center of each well under the microscope. Do not overlap follicles with each other.
    6. Identify follicles with posterior enzymatic activity (exhibiting green fluorescence at the posterior end) and segregate them to one side. See Video 2 for an example of identifying follicles with posterior enzymatic activity.
    7. Use the micromanager to capture bright-field, green-fluorescent, and red-fluorescent images.

      Video 2. Analysis of in situ zymography assay. Open images acquired at the end of each experiment (both bright-field and fluorescent images) in ImageJ. Count the total number of follicles using the multi-point tool in the bright-field image. In this example, there are a total of 29 follicles. Then, count the total number of follicles with posterior green fluorescence in the green-fluorescent image. Before taking the image, most of the follicles identified with enzymatic activity were segregated toward the top of the image. In this example, there are a total of 12 follicles with posterior green fluorescence; therefore, 41.4% of follicles had enzymatic activity.

Data analysis

Data are plotted as ‘Ruptured follicles (%)’. Each data point is one ‘group’ or well: count the total number of follicles in the well and the total number of ruptured follicles (see Video 3, Analysis of ex vivo follicle rupture assay). A follicle is categorized as ‘ruptured’ if > 80% of the oocyte is exposed. Divide the ruptured follicles by the total number of follicles to calculate ‘Ruptured follicles (%)’ (Deady et al., 2015).
For in situ zymography assay, data are plotted as ‘follicles with posterior green fluorescence (%)’. Count the total number of follicles with posterior green fluorescence and divide by the total number of follicles in the well (Deady et al., 2015). (See Video 2, Analysis of in situ zymography assay).

Video 3. Analysis of ex vivo follicle rupture assay. Open images acquired at the end of each experiment (both bright-field and fluorescent images) in ImageJ. Count the total number of follicles using the multi-point tool in the bright-field image. In this example, there are a total of 27 follicles. Then, count the total number of ruptured follicles in the fluorescent image. In this example, there are a total of 20 ruptured follicles; therefore, 74% of follicles ruptured in this example.

Notes

  1. Ruptured follicles (%) from wild-type flies will vary depending upon the fluorescent reporter used. For example, if a reporter only labels the most mature follicles (stage-14C; such as with the R47A04-Gal4 driver; Deady et al., 2017), one would expect to see ~80% of follicles ruptured at the end of the three-hour culture. In contrast, if all stage-14 follicles are labeled and isolated (such as with the R44E10-Gal4 driver; Deady et al., 2017), you would expect to observe ~50% of follicles ruptured. The reduced rupture rate is likely due to the isolation of slightly younger stage-14 follicles that are not competent to OA-induced follicle rupture (Deady et al., 2017).
  2. The ruptured follicles (%) in negative control groups (without octopamine) should be less than 10%. Greater than 10% ruptured follicles in negative controls will indicate that some of the isolated follicles have already initiated the rupture process (such as the breakdown of posterior follicle cells) before adding octopamine working solution. When setting up ex vivo follicle rupture, it is imperative that all follicles are completely intact. For an example of intact, partially ruptured, and ruptured follicles, see Figure 2.


    Figure 2. Examples of follicle cell layer coverage of oocyte. Scale bar is estimated 100 μm.

  3. Finish ‘Dissection and Isolation (Procedure C)’ within one hour.
  4. Minimize the amount of endogenous octopamine exposure that follicles receive during isolation. Don’t isolate follicles in the same media that the flies were dissected in; rather, use fresh Grace’s media.
  5. When transferring ~25-35 selected follicles to dry well, make sure they have enough media that they don’t dry out. Usually, the follicles are in ~15-30 μl culture media before adding the octopamine working solution.
  6. Thoroughly mix the octopamine working solution or other drugs before beginning the culture.
  7. During dissections, some follicle-cell layers can envelop the anterior egg chamber in the ovariole (see Figure 2E). Don’t select these egg chambers; they will not rupture.

Recipes

  1. Wet yeast paste
    Mix active dry yeast in distilled water. Consistency of the wet yeast paste should be semi-solid, all yeast granules should be dissolved yet yeast should not be watery
  2. Fly food (3 L)
    Yeast (g): 61
    Cornmeal (g): 163
    Molasses (ml): 203
    Agar (g): 22
    Water (ml): 3,000
    5% Tegosept (50 g Tegosept in 1,000 ml ethanol) (ml): 36
    Propionic acid (ml): 13
    Measure out all ingredients (except Tegosept and Propionic acid), mix thoroughly, and autoclave at 250 °F for 30 min. Once cooled to 70 °C, Tegosept and Propionic acid are added and mixed thoroughly before dispensing to the fly vials 
  3. Culture media mixture
    Add 1 ml of fetal bovine serum and 100 μl of penicillin-streptomycin (10,000 U/ml) to 9 ml of Grace’s media
  4. Octopamine stock solution (10 mM)
    Dissolve octopamine hydrochloride powder in distilled water to 10 mM. Freeze aliquots for up to six months
  5. Octopamine working solution (20 μM)
    For each well: 1 ml of culture media mixture + 2 μl of octopamine stock solution. Rock on the nutator for 1~3 min before use
  6. Fluorescein-conjugated DQTM gelatin stock solution (1 mg/ml)
    To make aqueous solution, dissolve 1 mg DQTM gelatin powder in 1 ml distilled water and store the solution in fridge covered with foil
  7. Gelatin working solution (25 μg/ml)
    For each well: 1 ml of culture media mixture + 25 μl of DG gelatin stock solution + 20 μl of octopamine stock solution. Rock on the nutator for 1~3 min before use

Acknowledgments

We thank current and past members of the Sun lab for suggestions and technical assistance. We are very grateful for Dr. Laurinda Jaffe for initial suggestion of ex vivo culture. JS is supported by the University of Connecticut Start-up fund, NIH/National Institute of Child Health and Human Development Grant R01-HD086175, and Bill and Melinda Gates Foundation. This protocol was implemented in previously published studies (Deady and Sun, 2015; Knapp and Sun, 2017; Deady et al., 2017) and used to identify genes required for ovulation in Drosophila.
The authors declare no conflict of interest.

References

  1. Aigaki, T., Fleischmann, I., Chen, P. S. and Kubli, E. (1991). Ectopic expression of sex peptide alters reproductive behavior of female D. melanogaster. Neuron 7(4): 557-563.
  2. Bloch Qazi, M. C., Heifetz, Y., Wolfner, M. F. (2003). The developments between gametogenesis and fertilization: ovulation and female sperm storage in drosophila melanogaster. Dev Biol 256 195-211.
  3. Cole, S. H., Carney, G. E., McClung, C. A., Willard, S. S., Taylor, B. J. and Hirsh, J. (2005). Two functional but noncomplementing Drosophila tyrosine decarboxylase genes: distinct roles for neural tyramine and octopamine in female fertility. J Biol Chem 280(15): 14948-14955.
  4. Deady, L. D., Shen, W., Mosure, S. A., Spradling, A. C. and Sun, J. (2015). Matrix metalloproteinase 2 is required for ovulation and corpus luteum formation in Drosophila. PLoS Genet 11(2): e1004989.
  5. Deady, L. D. and Sun, J. (2015). A follicle rupture assay reveals an essential role for follicular adrenergic signaling in Drosophila ovulation. PLoS Genet 11(10): e1005604.
  6. Deady, L. D., Li, W. and Sun, J. (2017). The zinc-finger transcription factor Hindsight regulates ovulation competency of Drosophila follicles. Elife 6: e29887.
  7. Edelstein, A. D., Tsuchida, M. A., Amodaj, N., Pinkard, H., Vale, R. D. and Stuurman, N. (2014). Advanced methods of microscope control using muManager software. J Biol Methods 1(2).
  8. Heifetz, Y., Lung, O., Frongillo, E. A., Jr. and Wolfner, M. F. (2000). The Drosophila seminal fluid protein Acp26Aa stimulates release of oocytes by the ovary. Curr Biol 10(2): 99-102.
  9. Knapp, E. and Sun, J. (2017). Steroid signaling in mature follicles is important for Drosophila ovulation. Proc Natl Acad Sci U S A 114(4): 699-704.
  10. Lee, H. G., Rohila, S. and Han, K. A. (2009). The octopamine receptor OAMB mediates ovulation via Ca2+/calmodulin-dependent protein kinase II in the Drosophila oviduct epithelium. PLoS One 4(3): e4716.
  11. Lee, H. G., Seong, C. S., Kim, Y. C., Davis, R. L. and Han, K. A. (2003). Octopamine receptor OAMB is required for ovulation in Drosophila melanogaster. Dev Biol 264(1): 179-190.
  12. Lim, J, Sabandal, P. R., Fernandez, A., Sabandal, J. M., Lee, H. G., Evans, P. and Han, K. A. (2014). The octopamine receptor Octβ2R regulates ovulation in Drosophila melanogaster. PLoS One 9: e104441.
  13. Monastirioti, M. (2003). Distinct octopamine cell population residing in the CNS abdominal ganglion controls ovulation in Drosophila melanogaster. Dev Biol 264(1): 38-49.
  14. Monastirioti, M., Linn, C. E., Jr. and White, K. (1996). Characterization of Drosophila tyramine beta-hydroxylase gene and isolation of mutant flies lacking octopamine. J Neurosci 16(12): 3900-3911.
  15. Pfeiffer, B. D., Jenett, A., Hammonds, A. S., Ngo, T.-T. B., Misra, S., Murphy, C., Scully, A., Carlson, J. W., Wan, K. H., Laverty, T. R., et al. (2008). Tools for neuroanatomy and neurogenetics in Drosophila. PNAS 105: 9715-9720.
  16. Schneider, C. A., Rasband, W. S. and Eliceiri, K. W. (2012). NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9(7): 671-675.
  17. Spradling, A. C. (1993). Developmental genetics of oogenesis. Cold Spring Harbor Laboratory Press.
  18. Sun, J. and Spradling, A. C. (2013). Ovulation in Drosophila is controlled by secretory cells of the female reproductive tract. Elife 2: e00415.

简介

排卵是从卵巢释放成熟卵母细胞的过程,对动物繁殖至关重要。 为了发生排卵过程,卵泡必须完全成熟并发信号通知卵巢破裂。 在哺乳动物和果蝇卵泡破裂期间,体细胞卵泡细胞被酶促降解以允许卵母细胞从卵泡释放。 在此,我们描述了我们在果蝇中新开发的离体卵泡破裂测定的详细方案,其代表允许直接量化卵泡响应排卵刺激的能力的第一测定 并破裂。 可以修改该测定以刺激用其他试剂(例如离子霉素)破裂或查询酶活性(原位酶谱法)。 此外,这种检测方法允许基因或药理学筛选鉴定果蝇中调节卵泡破裂的基因或小分子。

【背景】由于在直接显示和定量排卵事件方面的技术挑战,对果蝇中的排卵的研究在很大程度上受到限制。在过去的几十年中,多种间接方法已经有了局限性。研究果蝇排卵的第一种方法是推动女性的腹部,并记录是否有卵子从产卵器排出(Aigaki et al。,1991; Lee <等人,2003)。这种方法是粗略估计子宫内是否有排卵的卵子。第二种常见的测定方法是测量雌蝇交配后的产蛋能力。产卵过程复杂,涉及卵泡的发育和成熟,排卵,排卵通过输卵管的运输,选择合适的产卵基质和产卵(Spradling,1993; Bloch Qazi等人。,2003)。如果这些过程中的任何一个受到损害,则会导致产蛋率有缺陷。排卵受损的另一个指标是卵子保留表型(Monastirioti等人,1996; Monastirioti,2003; Cole等人,2005)。如果女性卵巢内有过多的成熟卵泡,则表示无排卵表型。然而,这可以直接由排卵缺陷引起,或间接由产卵过程中排卵下游的缺陷引起。另一方面,缺乏保留表型并不一定意味着缺乏排卵缺陷。用于研究排卵的第四种测定方法是检查生殖道中是否存在卵子(Heifetz et al。2000,Lee et al。2009; Lim 等人,,2014)。该测定法中的变化范围从量化排卵率的比例通过雌性在其下部输卵管/常见输卵管/子宫交配后的雌性百分比来定量(Lee等人,2009; Lim等人, ,2014年),以检查这些分开的区域随着时间的推移而分布的鸡蛋(Heifetz et al。 2000>)。然而,这些测定中的每一个都可能受卵子发生,排卵和产卵速度的影响。为了说明每个单独测定的所有可能的缺点,我们最近将雌性生殖道中的卵子保留测定,产蛋测定和卵子定位结合起来以估计卵子排卵的平均时间(排卵时间; Sun和Spradling,2013年; Deady et al。,2015年和2017年; Deady和Sun,2015年; Knapp和Sun,2017年)。然而,这种方法是乏味的,也依赖间接测量排卵。

我们最近在细胞水平上表征了排卵,并发现果蝇排卵涉及卵泡破裂过程。在排卵期间,后毛囊细胞激活基质金属蛋白酶2(Mmp2),后者降解后卵泡细胞,使被包裹的卵母细胞破裂进入输卵管(Deady et。,2015)。我们还发现这个过程是由卵泡细胞中的蘑菇体(Oamb)信号传导中的直接章鱼胺(octopamine,OA)和章巴胺受体启动的,整个过程可以在我们的体外培养系统中重现(Deady和Sun,2015)。我们将这种测定命名为离体卵泡破裂,其中从卵巢分离成熟卵泡并用OA刺激以诱导卵泡破裂。在三小时孵育结束时可报告毛囊破裂的百分比,这是对毛囊破裂的直接量化。该检测方法可以对果蝇中的卵泡破裂进行相对简单,高通量的检测,是遗传和药理筛选的理想选择。然而,这个实验是在体外完成的,并且应该使用上述的一些测定法来验证体内的结果。

关键字:果蝇, 排卵, 卵泡破裂, 章鱼胺, 原位酶谱法, 卵泡细胞

材料和试剂

  1. 500 ml Nalgene(Thermo Fisher Scientific TM,产品目录号:5700-0500)
  2. 铝箔
  3. 纸巾
  4. PYREX TM斑点板(9孔)(Corning,PYREX®,产品目录号:7220-85)
  5. 不锈钢针,0.25毫米,36毫米(泰德佩拉,目录号:13561)
  6. 塑料转移管,一次性,5.8毫升(Fisher Scientific,Fisherbrand,目录号:13-711-9CM)
  7. 1.5毫升Eppendorf管
  8. 格雷斯的媒体,与谷氨酰胺(杰纳西科学,目录号:25-516G)
  9. 活性干酵母(Genesee Scientific,目录号:62-103)
  10. 玉米面(Genesee Scientific,目录号:62-101)
  11. 糖蜜(Fisher Scientific,目录号:NC9109740)
    制造商:LabScientific,目录号:FLY-8008-16。
  12. 琼脂(Genesee Scientific,目录号:66-103)
  13. Tegosept(Genesee Scientific,目录号:20-258)
  14. 乙醇
  15. 丙酸(Sigma-Aldrich,目录号:P1386-1L)
  16. 胎牛血清(亚特兰大生物公司,目录号:S11150)
  17. 青霉素 - 链霉素(10,000U / ml)(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  18. 盐酸章鱼胺(Sigma-Aldrich,目录号:OO250-1G)
  19. 来自猪皮肤的荧光素 - 缀合的DQ TM明胶(Thermo Fisher Scientific,Invitrogen TM,目录号:D12054)
  20. 湿酵母酱(见食谱)
  21. 飞食物(见食谱)
  22. 培养基混合物(见食谱)
  23. 章鱼胺原液(见食谱)
  24. 章鱼胺工作溶液(见食谱)
  25. 荧光素结合的DQ TM明胶储备溶液(见食谱)
  26. 明胶工作解决方案(见食谱)

设备

  1. 杜蒙#5镊子(精细科学工具,目录号:11252-20)
  2. Pipetman试剂盒G系列(P20,P200,P1000)(Gilson,目录号:F167900)
  3. 用于荧光成像的模块化立体显微镜(Leica Microsystems,型号:Leica MZ10 F)
  4. sCMOS相机(PCO.4.2)
  5. 29°C湿度控制培养箱 - 设置为〜70%RH(Percival Scientific,型号:DR-36VL)
  6. CO 2罐,CD 50(Airgas,型号:CGA-320)
  7. 高压灭菌器
  8. Flypad(Genesee Scientific,目录号:59-114)
  9. Nutator(Fisher Scientific,型号:260100F)

软件

  1. ImageJ(NIH)(Schneider等人,2012)
  2. 微管理器(NIH)(Schneider等人,2012; Edelstein等人,2014)

程序

  1. 飞行饲养
    1. 遗传要求
      在成熟卵泡细胞(阶段14)中表达的荧光记者是分离具有完整卵泡细胞层的成熟卵泡所必需的。在我们的实验中,我们使用来自Janelia Gal4集合(Pfeiffer等人,2008)的R47A04-Gal4或R44E10-Gal4来驱动UAS-RFP报道分子特异性在14期卵泡中的表达细胞。
    2. 收集处女女性的正确基因型,并在29°C时龄5-6D。
    3. 在实验前用约1茶匙新鲜酵母酱补充食物2-3D。

  2. 实验准备
    1. 温格雷斯的媒体到室温(〜25°C)。
    2. 准备培养基混合物(见食谱)。
    3. 带铝箔的实用工具箱可以屏蔽内部光线。
    4. 在箱子底部添加湿纸巾以保持三小时孵育期间的湿度。

  3. 解剖和隔离(见图1)
    1. 确保最小化从解剖开始到解剖和隔离中最后一步的时间。
      在一小时内限制整个解剖和隔离
    2. 用1毫升格雷斯的培养基在PYREX点板上填一个孔(图1,'1.初步解剖')。


      图1. PYREX 9孔斑点板设置

    3. 在Grace的媒体中,使用镊子从八名女性中分析卵巢对。确保不要损害卵巢并保持完好。
      提示:抓住输卵管/子宫区域,而不是直接抓住卵巢,从腹部轻轻去除卵巢。
    4. 用〜1 ml格雷斯的培养基填充另一孔,并将所有卵巢对转移到这个新孔中(图1,'2。保持卵巢对')。
    5. 请参阅视频1.“解剖示例”
      1. 将卵巢对(每次〜两个)移动到1毫升格雷斯培养基的新孔中(图1,'3.分离卵泡')。
      2. 使用其中一个钳子来保持卵巢的前端,靠近germarium。
        使用其他镊子轻轻地分离卵巢后部靠近花萼的卵巢。
      3. 接下来,使用另一个镊子从后端轻轻挤压卵泡。这个过程是从周围的卵巢鞘释放卵泡。小心不要刺破毛囊。

      视频1

    6. 根据荧光记者鉴定成熟卵泡,并用针头制作一堆完整的成熟卵泡。
    7. 使用P20移液管将完整的成熟卵泡转移到装满约1ml培养基的新孔中(图1,'4.分离的,完整的S14卵泡池')。
      首先在培养基上涂抹移液器吸头,以防止滤泡粘在移液器吸头上。
    8. 重复步骤C4-C7,直到你有足够的卵泡,或直到约40分钟后才开始清扫。
    9. 清除池中任何年轻阶段的毛囊,并仔细检查池中的每个毛囊都保持完整。在荧光灯下观看时,年轻阶段的滤泡不应表达荧光记者,但可在明视野光下观看。
    10. 使用针将〜25-35个完整的卵泡堆积在一起。用P20移液管将培养基中的绒毛卵泡转移到新的孔中。这些滤泡不应该干燥 - 它们应该保留在它们转移的约20μl培养基中。重复此步骤,直到所有选定的卵泡以25-35的“组”形式分配到新的孔中,或者直到从初始解剖开始已经过去约50分钟为止(图1,'〜25-35完整的S14卵泡在〜20 μlCM')。
    11. 准备章鱼胺工作溶液(请参阅食谱)每个'组'为-1毫升。
    12. 向每个孔中加入1ml的章鱼胺工作溶液以刺激体外破裂或将1ml培养基作为阴性对照到每个孔中。
    13. 将PYREX斑点板放入一个贴有箔片的实用工具箱(在程序B中制备)中,盖上箱子,并将箱子放入29°C培养箱中3小时。

  4. 图像采集

    1. 三个小时后,从培养箱中取出盒子。
    2. 在显微镜下用针将毛囊堆积在每个孔的中心。不要将卵泡互相重叠。
    3. 使用微管理器捕捉明场和荧光图像。

  5. 修改原位酶谱图
    1. 按照步骤C1-C10制备分离的成熟卵泡。
    2. 准备明胶工作解决方案(见食谱)每个'团体'-1毫升。
    3. 向每个孔中加入1ml明胶工作溶液以刺激体外破裂并检测明胶酶活性。一个阴性对照应该是没有章鱼胺的明胶工作溶液。
    4. 在29°C孵育3小时后,通过吸出溶液并用新鲜培养基替换每个孔中的明胶工作溶液(注意不要从每个孔中去除滤泡)。
    5. 在显微镜下使用针将毛囊堆积在每个孔的中心。不要将卵泡互相重叠。
    6. 识别具有后酶活性的毛囊(在后端显示绿色荧光)并将它们分离到一侧。查看视频2,查找具有后酶活性的卵泡。
    7. 使用微管理器捕捉明场,绿色荧光和红色荧光图像。

      视频2

数据分析

数据绘制为'破裂的卵泡(%)'。每个数据点是一个“组”或好的数字:计数孔中毛囊的总数和破裂的毛囊总数(见视频3,分析 ex vivo 毛囊破裂试验)。如果&gt;毛囊被分类为“破裂” 80%的卵母细胞暴露。将破裂的卵泡除以卵泡总数来计算'破裂的卵泡(%)'(Deady et。,2015)。
为了进行原位分析,将数据绘制成“具有后绿色荧光的滤泡(%)”。计算后绿色荧光的卵泡总数除以井中卵泡总数(Deady et。,2015)。 (见视频2,原位分析法分析)。

视频3

笔记

  1. 来自野生型苍蝇的破裂卵泡(%)将根据所使用的荧光记者而变化。例如,如果一个记者只标记最成熟的卵泡(阶段14C;比如用R47A04-Gal4驱动; Deady et。,2017),人们希望看到〜80%的在三小时培养结束时卵泡破裂。相反,如果所有的14号卵泡都被标记和分离(例如用R44E10-Gal4驱动器; Deady等人,2017),你可以期望观察约50%的卵泡破裂。降低的破裂率很可能是由于分离了较年轻的14号卵泡而不能诱导OA诱导的卵泡破裂(Deady et al。,2017)。
  2. 阴性对照组(无章鱼胺)破裂的滤泡(%)应小于10%。阴性对照中破裂的卵泡超过10%将表明在添加章鱼胺工作溶液之前,一些分离的卵泡已经开始破裂过程(例如后卵泡细胞分解)。当建立离体卵泡破裂时,所有卵泡都完好无缺。有关完整,部分破裂和破裂卵泡的示例,请参见图2.


    图2.卵母细胞层覆盖卵母细胞的例子比例尺估计为100μm。


  3. 在一小时内完成解剖和隔离(程序C)'
  4. 尽量减少卵泡在分离期间接受的内源性章鱼胺暴露量。不要在与解剖苍蝇相同的媒介中分离卵泡;相反,请使用Grace的新媒体。
  5. 当转移〜25-35个选定的滤泡干燥时,确保它们有足够的介质不会变干。通常,在加入章鱼胺工作溶液之前,卵泡在〜15-30μl培养基中。
  6. 在开始培养前彻底混合章鱼胺工作溶液或其他药物。
  7. 在解剖过程中,一些卵泡细胞层可以包裹卵巢内的前卵腔(见图2E)。不要选择这些蛋室;他们不会破裂。

食谱

  1. 湿酵母酱
    将活性干酵母混入蒸馏水中。湿酵母酱的一致性应该是半固态的,所有的酵母颗粒应该溶解,但酵母不应该是水
  2. 飞食物(3升)
    酵母(g):61
    玉米面(g):163
    糖蜜(毫升):203
    琼脂(克):22
    水(毫升):3,000
    5%Tegosept(在1000ml乙醇中50g Tegosept)(ml):36
    丙酸(ml):13
    测量所有成分(Tegosept和丙酸除外),彻底混匀,并在250°F下高压灭菌30分钟。一旦冷却到70°C,Tegosept和Propionic酸被加入并充分混合,然后分配到飞蝇瓶中。
  3. 培养基混合物

    加入1 ml胎牛血清和100μl青霉素 - 链霉素(10,000 U / ml)至9 ml Grace培养基中
  4. 章鱼胺原液(10 mM)
    将盐酸章鱼胺粉末溶于蒸馏水至10 mM。
    冷冻等分试样长达六个月
  5. 章鱼胺工作溶液(20μM)
    对于每个孔:1ml培养基混合物+2μl章鱼胺储备溶液。使用前,在nutator上晃动1〜3分钟
  6. 荧光素缀合的DQ TM明胶储备溶液(1mg / ml)
    为了制成水溶液,将1毫克DQ TM明胶粉溶解在1毫升蒸馏水中并将溶液储存在覆盖有箔的冰箱中
  7. 明胶工作溶液(25μg/ ml)
    对于每个孔:1ml培养基混合物+25μlDG明胶储备溶液+20μl章鱼胺储备溶液。使用前,在nutator上晃动1〜3分钟

致谢

我们感谢Sun实验室的当前和过去的成员提供建议和技术援助。我们非常感谢Laurinda Jaffe博士最初提出的 ex vivo 文化。 JS由康涅狄格大学创业基金,美国国立卫生研究院/国家儿童健康研究所和人类发展基金R01-HD086175以及比尔和梅林达盖茨基金会提供支持。该协议在以前发表的研究中得到实施(Deady和Sun,2015; Knapp和Sun,2017; Deady等人,2017),并用于鉴定果蝇中排卵所需的基因。
作者宣称没有利益冲突。

参考

  1. Aigaki,T.,Fleischmann,I.,Chen,P.S。和Kubli,E。(1991)。 性肽的异位表达会改变女性D的生殖行为。 melanogaster 。 Neuron 7(4):557-563。
  2. Bloch Qazi,M.C.,Heifetz,Y.,Wolfner,M.F。(2003)。 配子发生与受精之间的进展:果蝇排卵和雌性精子储存 Dev Biol 256 195-211。
  3. Cole,S.H.,Carney,G.E.,McClung,C.A.,Willard,S.S.,Taylor,B.J。和Hirsh,J。(2005)。 两种功能性但不互补的果蝇酪氨酸脱羧酶基因:神经酪胺的不同作用和女性生育能力的章鱼胺。 J Biol Chem 280(15):14948-14955。
  4. Deady,L.D.,Shen,W.,Mosure,S.A。,Spradling,A.C。和Sun,J。(2015)。 基质金属蛋白酶2是果蝇中排卵和黄体形成所必需的。 PLoS Genet 11(2):e1004989。
  5. Deady,L.D。和Sun,J。(2015)。 卵泡破裂试验揭示了果蝇中滤泡肾上腺素能信号的重要作用排卵。 Genet 11(10):e1005604。
  6. Deady,L. D.,Li,W。和Sun,J。(2017)。 锌指转录因子Hindsight调节果蝇卵泡的排卵能力。 Elife 6:e29887。
  7. Edelstein,A.D.,Tsuchida,M.A.,Amodaj,N.,Pinkard,H.,Vale,R.D。和Stuurman,N.(2014)。 使用muManager软件进行显微镜控制的先进方法 J Biol方法 1(2)。
  8. Heifetz,Y.,Lung,O.,Frongillo,E.A.,Jr.和Wolfner,M.F。(2000)。 果蝇精液蛋白Acp26Aa刺激卵巢释放卵母细胞。 Curr Biol 10(2):99-102。
  9. Knapp,E。和Sun,J。(2017)。 成熟卵泡中的类固醇信号传导对果蝇排卵非常重要。。美国国家科学院院刊 114(4):699-704。
  10. Lee,H.G.,Rohila,S.and Han,K.A。(2009)。 章鱼胺受体OAMB通过Ca2 + /钙调蛋白依赖介导排卵蛋白激酶II在果蝇输卵管上皮中的表达。 PLoS One 4(3):e4716。
  11. Lee,H.G.,Seong,C.S.,Kim,Y.C。,Davis,R.L。和Han,K.A。(2003)。 Octopamine受体OAMB是果蝇排卵所必需的。 > Dev Biol 264(1):179-190。
  12. Lim,J,Sabandal,P.R.,Fernandez,A.,Sabandal,J.M.,Lee,H.G.,Evans,P.and Han,K.A。(2014)。 octopamine受体Octβ2R调节果蝇的排卵。 PLoS One 9:e104441。
  13. Monastirioti,M.(2003)。 居住在中枢神经系统腹部神经节中的不同章鱼胺细胞群控制果蝇中的排卵。 Biol 264(1):38-49。
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Copyright Knapp et al. 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. Knapp, E. M., Deady, L. D. and Sun, J. (2018). Ex vivo Follicle Rupture and in situ Zymography in Drosophila. Bio-protocol 8(10): e2846. DOI: 10.21769/BioProtoc.2846.
  2. Deady, L. D., Li, W. and Sun, J. (2017). The zinc-finger transcription factor Hindsight regulates ovulation competency of Drosophila follicles. Elife 6: e29887.
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