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Feb 2020

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Analysis of Caenorhabditis elegans Sperm Number, Size, Activation, and Mitochondrial Content
秀丽隐杆线虫精子数量、大小、活化及线粒体含量分析   

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Abstract

Infertility is a widespread and often unexplained issue. Studying reproduction using C. elegans males offers insight into the influence of individual factors on male fertility in humans. We have created a collection of protocols to assess several aspects of C. elegans sperm quality, including number, size, rate of activation, and mitochondrial morphology. Studying sperm biology in a model system such as C. elegans allows access to the wealth of resources and techniques that have been optimized for that organism while providing valuable biological information that may be applicable to other systems.


Graphic abstract:



Flowchart depicting the preparation of C. elegans males and subsequent sperm quality assays

Keywords: C. elegans (秀丽隐杆线虫), Fertility (生育力), Sperm quality (精子质量), Spermatogenesis (精子形成), Mitochondria (线粒体)

Background

Approximately 8% of couples worldwide suffer from infertility (World Health Organization, 1987), and 30% of those cases have unknown causes (Practice Committee of the American Society for Reproductive, 2006). The wide variety of factors that affect male fertility, both genetic and environmental (Oliva et al., 2001; Huynh et al., 2002), make studies in humans susceptible to confounding influences. Model systems can be useful in investigating the influence of single factors on various aspects of fertility. Caenorhabditis elegans is a genetically tractable organism with a defined number of cells and germ cells that can be examined phenotypically.


Studies of mammalian mitochondrial function (including mitochondrial morphology, mitochondrial genome and copy number, mitochondrial protein levels, and enzyme activity of the electron transport chain complexes) have demonstrated a link between mitochondrial health and sperm function (Amaral et al., 2013). Additionally, several mitochondrial mutants negatively impact C. elegans fertility, although the exact mechanism of action of these pathways is unknown (Jonassen et al., 2002; Grad and Lemire, 2004). Recent work from our lab has demonstrated that mafr-1, a regulator of RNA polymerase III (Hammerquist and Curran, 2020), and alh-6 and prdh-1, enzymes in the mitochondrial proline catabolism pathway (Yen et al., 2020; Yen and Curran, 2021), are required for proper sperm function in C. elegans, and these roles may be conserved in mammalian models (Bonhoure et al., 2015; Yen and Curran, 2021).


We recently developed and published methods to assess various metrics of spermatid quality in C. elegans (Hammerquist and Curran, 2020; Yen and Curran, 2020; Yen et al., 2020). Some of the methods we present, albeit with added details and refined techniques, are largely unchanged from their original publication decades ago (Ward et al., 1983; Shakes and Ward, 1989; LaMunyon and Ward, 1998), whereas others, specifically the quantification of spermatid mitochondrial fusion, were first developed in our lab (Yen et al., 2020). Here, we provide detailed protocols for a comprehensive set of assays that measure the quality and mitochondrial health of spermatid.


Materials and Reagents

  1. Microscope slides (VWR, catalog number: 48312-004)

  2. Sterile 6 cm Petri dishes (VWR, catalog number: 25373-085)

  3. Platinum wire (Tritech Research, catalog number: PT-9010)

  4. Eyelash brush (consisting of a human eyelash attached to a Pasteur pipette, generic, with tape, generic)

  5. Microscope slide cover slips (VWR, catalog number: 48366-227)

  6. PAP pen (Liquid Blocker, catalog number: Z377821)

  7. Plastic or glass container that can be sealed (Glasslock, catalog number: OCRT-048)

  8. Paper towels (generic)

  9. Parafilm (Sigma-Aldrich, catalog number: P7543)

  10. 25G needles (BD, catalog number: 305125) or scalpel (Technocut, catalog number: 6008T-10)

  11. 750 ml centrifuge bottles that can be sterilized by autoclaving (generic)

  12. 2-200 μl micropipette tips (Genesee Scientific, catalog number: 24-151RL)

  13. E. coli OP50-1 (available from Caenorhabditis Genetics Center)

  14. Worm strains (available from Caenorhabditis Genetics Center)

  15. Sodium Chloride (Fisher Scientific, catalog number: 02-004-047)

  16. Peptone (BD, catalog number: 211820)

  17. Bacto Agar (BD, catalog number: 214040)

  18. Cholesterol (Sigma-Aldrich, catalog number: C8667)

  19. Ethanol (VWR, catalog number: 89125-172)

  20. Calcium Chloride (Sigma-Aldrich, catalog number: C3881)

  21. Magnesium Sulfate (Sigma-Aldrich, catalog number: M2773)

  22. Potassium Phosphate dibasic (Sigma-Aldrich, catalog number: P5504)

  23. Potassium Phosphate monobasic (Sigma-Aldrich, catalog number: P0662)

  24. Streptomycin sulfate (Sigma-Aldrich, catalog number: S6501)

  25. LB powder (Teknova, catalog number: L9315)

  26. HEPES (J.T. Baker, catalog number: 4018-04)

  27. Potassium Chloride (Sigma-Aldrich, catalog number: P3911)

  28. BSA (Sigma-Aldrich, catalog number: A9647)

  29. Dextrose (J.T. Baker, catalog number: JT1919)

  30. Sodium Phosphate dibasic (Sigma-Aldrich, catalog number: S9763)

  31. Potassium Chloride (Sigma-Aldrich, catalog number: P3911)

  32. Triton X-100 (Sigma-Aldrich, catalog number: X100-100 ml)

  33. Isopropanol (BDH, catalog number: BDH1133)

  34. DAPI (Sigma-Aldrich, catalog number: D9542)

  35. Petroleum jelly (Vaseline)

  36. Nail polish (clear, generic)

  37. Immersion oil for microscope (Fisher Scientific, catalog number: 12-624-66B)

  38. Vectashield mounting medium (VWR, catalog number: H-1000)

  39. Pronase (Millipore Sigma, catalog number: 53702)

  40. MitoTrackerTM Red CMXRos (Thermo Fisher, catalog number: M7512)

  41. MitoProbe TM JC-1 Assay Kit (Thermo Fisher, catalog number: M34152)

  42. Nematode Growth Medium (NGM) plates (see Recipes)

  43. Phosphate buffered saline (PBS) (see Recipes)

  44. PBS + 0.01% Triton X-100 (PBST) (see Recipes)

  45. SM Buffer (dextrose) (see Recipes)

  46. SM Buffer (BSA) (see Recipes)

  47. M9 (see Recipes)

  48. DAPI stock solution (see Recipes)

  49. Pronase working solution (see Recipes)

  50. MitoTracker stock solution (see Recipes)

  51. JC-1 stock solution (see Recipes)

Equipment

  1. 2-20 μl micropipette (Gilson FA10003M)

  2. 20-200 μl micropipette (Gilson FA10005M)

  3. Scale (generic)

  4. pH meter (generic)

  5. Microcentrifuge for 1.5 ml tubes (Eppendorf, model: 5430)

  6. Refrigerated centrifuge with swing bucket rotor for 750 ml centrifuge bottles (Beckman Coulter, model: Allegra X-15R)

  7. Tube rotator (Thermo Scientific, catalog number: 88881001)

  8. Bunsen burner (generic)

  9. Dissecting microscope (Nikon SMZ 800 with fiber optic diascopic illumination stand)

  10. Compound microscope with DIC, DAPI, GFP, and RFP filters, and 10×, 40-63×, and 100× objectives (Zeiss, model: AxioScope5)

  11. Color camera (Zeiss AxioCam MRm)

  12. Worm incubator (Generic, maintain at 20 °C)

Software

  1. Imaging software

    Dependent on the microscope used (in this case, we used a Zeiss AxioScope and the associated ZEN imaging software)

  2. ImageJ (available from NIH)

  3. Data analysis software

    We use GraphPad prism, although any software capable of t-test will suffice. ANOVA may be helpful if comparing multiple conditions but not strictly necessary.

Procedure

  1. Prepare slides (to be used in D. Sperm size assay; E. Sperm activation assay; F. Qualitative mitochondrial morphology assay; and G. Quantitative mitochondrial fusion assay).

    1. Using a PAP pen, draw circles of 1-1.5 cm in diameter on the microscope slides.

      Note: One standard slide will easily fit two grease circles, with room to cover each individually (when using 22 × 22 mm square cover slips).

    2. Allow to dry overnight (or > 2 h in lamellar flow hood).


  2. Prepare males

    1. Maintain the growth of males in the strains needed for the assay.

      Notes:

      1. Thirty to forty males are needed for sperm number assay; at least five males are needed per replicate of sperm size, activation, and mitochondrial assays. This number is easily obtained from a mixed population of males and hermaphrodites. To ensure the propagation of males in the strains used, transfer young adult males and hermaphrodites (3:1 ratio) to fresh plates 4-5 days prior to picking males for the assay.

      2. If mating ability and/or male fertility is impaired, males should be mated to hermaphrodites (3:1 ratio) overnight on NGM plates seeded with 20 μl of 25× OP50-1 (see Recipe 1i Note for further detail) and transferred to stock NGM plates. The smaller food spots increase the frequency of encounters between males and hermaphrodites, thus increasing the likelihood of successful mating. Mate animals 4-5 days prior to picking males for the assay.

      3. If larger numbers of animals are desired, populations can be synchronized by alkaline hypochlorite treatment of a mixed population of males and hermaphrodites as described before (Yen and Curran, 2020). To ensure you have enough males, only use populations with approximately 50% of males.

    2. Pick larval stage 4 (L4) virgin males to stock NGM plates free of hermaphrodites on the day prior to assay (consult WormAtlas (Altun et al., 2021) for further information on identifying L4 males).

      Notes:

      1. Picking L4 males ensures that premature activation does not occur via mating prior to the assays. Males should be prepared in this way for all assays, except for qualitative mitochondrial morphology, for which the MitoTracker dye should be applied to food prior to transferring males to the plates (see Procedure F).

      2. Males tend to crawl off plates in search of hermaphrodites if isolated for too long; if this becomes a problem, some loss of males can be prevented by allowing hermaphrodites (preferably gravid, unmated ones) to crawl on seeded NGM to scent it with pheromones and removing them before transferring males to plate.

    3. Incubate for 18-24 h at 20 °C.


  3. Sperm number assay

    1. Wash male worms from isolation plate with PBS + 0.01% Triton X-100 (PBST) in a 1.5 ml tube.

    2. Centrifuge for 1 min at 560 × g. Remove supernatant to 0.1 ml (use marks on the tube as a guide).

    3. Wash 1-2 additional times with PBST.

      Note: These washes remove E. coli from the worms to be stained. Wash until the supernatant removed is clear (and not cloudy with bacteria). Do not wash beyond this point. Additional washes increase the likelihood of losing worms while aspirating the supernatant.

    4. Centrifuge for 1 min at 560 × g. Remove supernatant to 0.1 ml.

    5. Add 600 μl of 40% isopropanol and rotate for 3 min at room temperature to fix worms.

    6. While worms are fixing, make staining solution: dilute 1 mg/ml DAPI stock solution to 10 μg/ml in 40% isopropanol (10 μl DAPI per 1 ml 40% isopropanol).

    7. Centrifuge for 1 min at 560 × g. Remove supernatant to 0.1 ml.

    8. Add 600 μl of staining solution and incubate in the dark for 5 min.

    9. Centrifuge for 1 min at 560 × g. Remove supernatant to 0.1 ml.

    10. Add 600 μl PBST and incubate in the dark for 30 min to destain.

    11. Mount samples on microscope slides (regular slides, not those prepared in Procedure A) with Vectashield mounting medium.

    12. Cover with a cover slip and seal the edges with nail polish. Allow to dry (in the dark) for 5-10 min.

    13. Image at 40-63× with DIC and DAPI filters, ensuring all spermatids are visible in each worm.

      Note: Spermatids should be restricted to the seminal vesicle in male worms and can be distinguished from other germ cells by their compact nuclei.

    14. Collect z-stacks of each seminal vesicle, ensuring all spermatids are captured (use 40× if needed to zoom out to include all spermatids). A representative image is shown in Figure 1.

      Note: Set bounds at upper and lower edges of the seminal vesicle (where the first spermatid is visible on each side of the seminal vesicle) and image at a z-plane distance of 0.25 microns. This distance is determined by the auto settings of the Zeiss imaging software.



      Figure 1. DAPI-stained spermatids in the seminal vesicle. The worm is positioned with the anterior region to the left and posterior to the right. The seminal vesicle is outlined in yellow. Scale bar, 20 μm.


  4. Sperm size assay

    1. Clean worms to get rid of E. coli.

      Cleaning can be done in one of two ways. For the first method, pipet two drops of ~15 μl M9 onto a Petri dish lid and use a platinum wire worm pick to transfer 5 day one adult virgin males to a drop of M9. Using an eyelash brush, transfer the worms into the second M9 drop before moving them into the SM buffer on the prepared slide. For the second method, use a platinum pick to transfer >5 day one adult virgin males to an unseeded NGM plate and allow them to crawl from the food spot. Then use a clean platinum pick (without bacteria) to transfer 5 males from the unseeded plate to the SM buffer on the prepared slide. Carefully move the worms individually at each transfer step. Picking up worms without food requires practice and is done in a scooping motion where the pick goes below the worm.

    2. Pipet 35 μl of SM buffer containing dextrose into the grease circle on the prepared slide (from Procedure A).

    3. Transfer 5 clean virgin males to the SM buffer on the slide using either an eyelash brush or a platinum pick, depending on how the animals were cleaned (see Step D1).

    4. Under a dissecting microscope, dissect worms to release the sperm.

      1. Using a scalpel or two 25-gauge needles (slide flat surfaces of needles across each other mimicking scissor blades), slice males between the midpoint and tail (approximately 1/3 body length from the tail).

        Note: Move rapidly as dissected sperm may be sensitive to changes in salt concentration and/or pH resulting from evaporation.

      2. Sterilize the scalpel or needles between samples by dipping in ≥ 70% ethanol and passing through a Bunsen burner flame. Allow to cool before use.

    5. Cover dissected spermatids with a cover slip.

    6. Seal with nail polish.

    7. Image with a 100× objective using a DIC filter. A representative image is shown in Figure 2.

      1. Complete imaging as quickly as possible because spontaneous activation has been observed when isolated spermatids are left for prolonged periods. In experienced hands, this should take no more than 5 min.

      2. Score only spherical cells and at least 100 per replicate.

        Note: Typically, 100 spermatids are easy to obtain from one slide of five dissected day one adult males. This is attainable from imaging approximately 5-10 fields. The number of fields imaged depends on how the dissection spreads them on the slides. It may help to keep a rough mental count on the number of spermatids while imaging; always take a few extra images.



      Figure 2. DIC image of isolated spermatids for size analysis . One spermatid has been outlined (in yellow) in ImageJ for size analysis. Scale bar, 10 μm.


  5. Sperm activation assay

    1. Prepare a humid chamber (Figure 3)

      1. Place a wet paper towel on the bottom of a lidded container. Cover with a sheet of parafilm to create a dry surface to place slides on.

      2. Cover with the lid.



      Figure 3. DIY humid chamber in open (A) and closed (B) configurations


    2. Prepare the Pronase working solution (see Recipes) and SM containing BSA and 400 ng/μl of Pronase.

      After the Pronase working solution is completely dissolved, dilute 4 μl in 96 μl of SM buffer containing BSA.

    3. Clean worms to remove E. coli (see Procedure D for details).

    4. Pipet 35 μl of SM buffer containing BSA into the grease circle on the prepared slide (from Procedure A).

      Note: Activation assays are done using SM buffer containing BSA rather than Dextrose. We have tested with both supplements and noted that BSA is needed for activation.

    5. Transfer 5 clean virgin males to the SM buffer on the slide using either an eyelash brush or a platinum pick, depending on how animals were cleaned (see Step D1a).

    6. Under a dissecting microscope, dissect worms to release the sperm (see Procedure D for instructions).

    7. After dissecting the final male, add 35 μl of SM buffer containing BSA and 400 ng/μl Pronase.

    8. Incubate in the humid chamber (covered) for 15 min.

      Note: In our hands, 15 min activates approximately 80% of WT spermatids, but the activation is sensitive to multiple factors, including handling time, temperature, and freshness of buffer components. The incubation length may need to be determined empirically on an individual basis.

    9. During the incubation, prepare the cover slip.

      1. Using forceps or a pipette tip, create a thin line of petroleum jelly around the edges of the coverslip.

        Note: We have found that sealing the slides with nail polish affects activation and thus use only petroleum jelly to seal slides for this assay.

    1. After 15 min, cover the dissected animals with the prepared cover slip. Do not seal further.

    2. Image with a 100× objective using a DIC filter. A representative image is shown in Figure 4.

      Notes:

      1. Because sperm may be in different focal planes, and pseudopods of activated spermatids may be difficult to see in a single image, we recommend imaging each field of view in two different focal planes to aid in scoring (this distance need not be precisely measured). The second image serves as an aid in determining the activation status of spermatids that are unclear in the first one. Often, the movement of pseudopods in the time between capturing the images also helps reveal details that might not be evident in a single image.

      2. Because the cover slip is adhered to the slide using only surface tension, changing objectives after oil immersion may cause it to shift or come off completely. For this reason, examine the slide using a lower magnification (non-oil immersion) objective before imaging. Do not return to a lower power objective after immersion oil is applied.


      1. Ensure all imaging is complete within the first 5 min after the incubation is complete (20 min after adding SM/Pronase solution).

      2. Score at least 100 spermatids per replicate.



      Figure 4. DIC image of sperm isolated for activation assays at various states of activation, showing spike (black arrowhead), lobed (black arrow), and full pseudopod (white arrowhead) projections. Please refer to Figure 2 for inactive (round) spermatids. Scale bar, 10 μm.


  6. Qualitative mitochondrial morphology assay

    1. Dilute the MitoTracker stock solution (1 mM in DMSO) to 100 μM in M9.

    2. Add 50 μl of working MitoTracker solution to the food spot of NGM plate seeded with 50 μl of 25× concentrated OP50 and let dry.

    3. Transfer L4 males to the prepared plate (from the previous step) using either an eyelash brush or a platinum pick, depending on how animals were cleaned (see Step D1).

    4. Incubate for 18-24 h at 20 °C.

    5. Clean worms to remove E. coli (see Procedure D for details).

    6. Pipet 35 μl of SM buffer containing dextrose into the grease circle on the prepared slide (from Procedure A).

    7. Under a dissecting microscope, dissect worms to release the sperm (see Procedure D for instructions).

    8. Cover the dissected spermatids with a cover slip.

    9. Seal with nail polish.

    10. Image with a 100× objective using DIC and AlexaFluor594 filters (RFP channels should also work). Representative images are shown in Figure 5.

      1. Ensure imaging is complete within 10 min.

      2. Score only spherical cells.

      3. Score at least 100 spermatids per replicate.



      Figure 5. MitoTracker-stained spermatids in DIC (A) and AF594 (B) channels. Scale bar, 2 μm.


  7. Quantitative mitochondrial fusion assay

    1. Prepare a humid chamber (see Procedure E for details).

    2. Clean worms to remove E. coli (see Procedure D for details).

    3. Prepare staining solution.

      1. Add 8 μl of 1.5 mM JC-1 stock to 792 μl of SM containing BSA (15 μM final concentration).

    1. Pipet 35 μl of SM buffer containing BSA with JC-1 into the grease circle on the prepared slide (from Procedure A).

    2. Transfer 5 cleaned virgin males to the SM buffer on the slide using either an eyelash brush or a platinum pick, depending on how animals were cleaned (see Step D1a).

    3. Under a dissecting microscope, dissect worms to release the sperm (see Procedure D for instructions).

    4. After dissecting the final male, add 25 μl of SM buffer containing BSA and JC-1.

    5. Incubate in the humid chamber (covered) for 10 min.

    6. Wash three times with 100 μl of SM buffer containing BSA (no JC-1).

      1. Be careful to avoid completely removing the liquid from dissected spermatids, as this will affect spermatid physiology (and therefore mitochondrial phenotypes).

      2. Be careful to avoid overflowing the grease circle and losing the sample. If necessary, use smaller wash volumes (80-90 μl).

    1. Cover the dissected animals with a cover slip and seal with nail polish.

    2. Image with a 100× objective using AlexaFluor488 (Green) and AlexaFluor546 (Red) channels. Representative images are shown in Figure 6.

      Note: JC-1 is a mitochondria-specific dye that reflects changes in membrane potential. The ratio of red fluorescence (high membrane potential mitochondria species) to green fluorescence (low membrane potential mitochondria species) is a commonly used metric of mitochondrial health (Sivandzade et al., 2019).

      1. Collect z-stacks of spermatids to be scored.

        Note: Collect 8-9 images with a spacing of 0.8 microns in the z-plane.

      2. Ensure imaging is complete within 10 min, as spermatids are alive (not fixed) and can undergo mitochondrial changes.

      3. Score only spherical cells.

      4. Score at least 40 spermatids per replicate.



      Figure 6. JC-1-stained spermatids from wild type (A-B) and alh-6 mutant (C-D) animals, showing mostly spherical and oblong mitochondria, respectively. (B and D) ImageJ detection of JC-1 stained sperm mitochondria. Spermatid Outlines are marked with white dashed lines. Scale bar, 1 μm. This image was previously published in Yen et al. (2020).

    Data analysis

    1. Sperm number assay

      1. Deconvolve z-stacks.

      2. Count spermatid nuclei in z-stacks.

        Note: To avoid counting the same nuclei more than once or losing track of the ones counted, divide the seminal vesicle into multiple sections by drawing lines and counting one section at a time. For reference, in a 40× stack of images for one seminal vesicle, we selected 50-80 sections. The number of sections will depend on how the worm lays and how much mounting medium is between the cover slip and the slide (the smaller the volume, the flatter the worms may be, and fewer z-stacks will be needed to acquire the image, whereas more counting sections may be needed).

      3. Each replicate should consist of at least 10 animals.

      4. At least three separate replicates should be completed. Statistical analysis should be performed using the Student’s t-test.

        Note: Data should be presented as the average sperm number per animal (a mean value of 30+ individual counts across three or more separate replicates).


    2. Size assay

      1. Score only non-activated, circular spermatids that are in focus.

      2. Each replicate should consist of at least 100 useable spermatids.

      3. Using ImageJ’s oval tool, hold shift to ensure circularity and outline and measure the area of spermatids that meet the inclusion criteria.

      4. At least three separate replicates should be completed. Statistical analysis should be performed using the Student’s t-test.

        Note: Data should be presented as the average spermatid size (a mean value of 300+ individual spermatids across three or more separate replicates).


    3. Activation assay

      1. Using images of both focal planes for each field of view, score individual spermatids as activated (with pseudopod) or not activated (no pseudopod, including cells that are in the process of becoming activated with spiked or lobed projections).

      2. Each replicate should consist of at least 100 scored spermatids.

      3. For each condition, calculate the percentage of activated spermatids (out of total scored).

      4. At least three separate replicates should be completed.

      5. Calculate the average activation percentage per replicate and compare the samples using the Student’s t-test.

        Note: Data should be presented as the average activation percentage (a mean value of three or more percentages values, one per replicate).


    4. Qualitative mitochondrial morphology assay

      This is a qualitative assay.


    5. Quantitative mitochondrial fusion assay

      1. Score z-stacks taken with the AlexaFluor546 filter. Use z-stacks to determine and score whether mitochondria are spherical (not fused) or oblong (fused).

      2. Each replicate should consist of at least 40 individual spermatids.

      3. For each condition, calculate the percentage of fused (out of total scored) mitochondria.

      4. At least three separate replicates should be completed.

      5. Compare data using the Student’s t-test.

        Notes:

        1. Data should be presented as the average percentage of fused mitochondria per spermatid (a mean value of 90+ percentages across three or more separate replicates).

        2. Changes in membrane potential can be determined using the fluorescence ratio of red to green channels using the JC-1 dye. ImageJ can be used for fluorescence analysis.

    Recipes

    1. Nematode Growth Medium (NGM) plates

      1. Prepare stock solutions of 1 M MgSO4, 5 mg/ml cholesterol, 1 M KH2PO4, 1 M CaCl2, and 2.5% (w/v) streptomycin.

        1. Dissolve 120.366 g of MgSO4 per 1 L water (1 M). Filter sterilize.

        2. Dissolve 5 g of cholesterol per 1 L 100% ethanol (5 mg/ml). Store at 4 °C.

        3. Dissolve 136.086 g of KH2PO4 per 1 L water (1 M). Filter sterilize.

        4. Dissolve 110.98 g of CaCl2 per 1 L water (1 M). Filter sterilize.

        5. Dissolve 25 g of streptomycin sulfate per 1 L water (2.5%). Filter sterilize. Store at 4 °C.

      2. Add 3 g of NaCl, 17 g of agar, 2.5 g of peptone, and 950 ml of water to a glass flask. Include a stir bar.

      3. Cover with foil and autoclave to sterilize and dissolve agar.

      4. Cool, with stirring, to 55-60 °C.

      5. Add 1 ml of 1 M MgSO4, 1 ml of 5 mg/ml cholesterol, 25 ml of 1 M KH2PO4, 1 ml of 1 M CaCl2, and 7.5 ml of 2.5% streptomycin.

      6. Stir 5 min.

      7. Dispense 11 ml into 6 cm Petri dishes using sterile technique.

      8. Allow to solidify overnight.

      9. Seed stock NGM plates with 250 μl of OP50-1 overnight culture (grown in LB + streptomycin, without shaking).

        Note: Mitotracker staining (Qualitative mitochondrial morphology assay) requires 25× concentrated bacteria. To make this, inoculate OP50-1 in 500 mL of LB + streptomycin. Grow with shaking, overnight at 37 °C. Centrifuge 20 min 5,000 × g at 4 °C (repeat as necessary to pellet bacteria from entire culture). Resuspend in sterile M9. Bring volume to 20 ml. Store at 4 °C for up to 6 months.

      10. Allow plates to dry (covered) 2-3 days before use.

    2. Phosphate buffered saline (PBS)

      1. Add 26.5 g of Na2HPO4·7H2O, 80 g of NaCl, 2 g of KCl, and 2 g of KH2PO4 to a glass bottle.

      2. Bring volume to 1 L.

      3. Sterilize by autoclaving.

    3. PBS + 0.01% Triton X-100 (PBST)

      Using a cut pipette tip (to broaden opening), add 100 μl of Triton X-100 to 1 L sterile PBS. Mix well (do not shake).

    4. SM Buffer (dextrose)

      1. Prepare stock solutions of 1 M HEPES, 1 M NaCl, 1 M KCl, 1 M CaCl2, 1 M MgSO4, and 1 M dextrose.

        1. Dissolve 238.3 g of HEPES per 1 L water (1 M). Filter sterilize.

          Although not necessary at this point, adjusting the pH to 7.8 before bringing solution to the final volume and sterilizing will expedite step 4c.

        2. Dissolve 58.4 g of NaCl per 1 L water (1 M). Filter or autoclave to sterilize.

        3. Dissolve 74.6 g of KCl per 1 L water (1 M). Filter or autoclave to sterilize.

        4. Dissolve 110.98 g of CaCl2 per 1 L water (1 M). Filter or autoclave to sterilize.

        5. Dissolve 120.366 g of MgSO4 per 1 L water (1 M). Filter or autoclave to sterilize.

        6. Dissolve 180.2 g of dextrose per 1 L water (1 M). Filter or autoclave to sterilize.

      2. Combine 2.5 ml of 1 M HEPES, 2.5 ml of 1 M NaCl, 1.25 ml of 1 M KCl, 250 μl of 1 M CaCl2, 50 μl of 1 M MgSO4, 500 μl of 1 M dextrose, and approximately 20 ml of water.

      3. Adjust pH to 7.8.

      4. Bring volume to 50 ml. Filter sterilize.

      5. Store at 4 °C. Allow aliquots to come to RT before use.

    5. SM Buffer (BSA)

      1. Prepare stock solutions of 1 M HEPES, 1 M NaCl, 1 M KCl, 1 M CaCl2, 1 M MgSO4, and 5% (w/v) BSA.

        1. Dissolve 238.3 g of HEPES per 1 L water (1 M). Filter sterilize.

          Although not necessary, adjusting the pH to 7.8 before bringing the solution to the final volume and sterilizing will expedite step 5c.

        2. Dissolve 58.4 g of NaCl per 1 L water (1 M). Filter or autoclave to sterilize.

        3. Dissolve 74.6 g of KCl per 1 L water (1 M). Filter or autoclave to sterilize.

        4. Dissolve 110.98 g of CaCl2 per 1 L water (1 M). Filter or autoclave to sterilize.

        5. Dissolve 120.366 g of MgSO4 per 1 L water (1 M). Filter or autoclave to sterilize.

        6. Dissolve 2.5 g of BSA per 50 ml water (5% w/v). Filter to sterilize. Store at 4 °C.

      2. Combine 2.5 ml of 1 M HEPES, 2.5 ml of 1 M NaCl, 1.25 ml of 1 M KCl, 250 μl of 1 M CaCl2, 50 μl of 1 M MgSO4, 1 ml of 5% (w/v) BSA, and approximately 20 ml of water.

      3. Adjust pH to 7.8.

      4. Bring volume to 50 ml. Filter sterilize.

      5. Store at 4 °C. Allow aliquots to come to RT before use.

    6. M9

      1. Add 30 g of KH2PO4, 60 g of Na2HPO4, 50 g of NaCl, and 120 mg of MgSO4 to a glass bottle. Bring volume to 1 L.

      2. Sterilize by autoclaving.

        Precipitation of calcium salts may occur after autoclaving. Re-dissolving may take several days. Agitation helps.

    7. DAPI stock solution

      Dissolve 1 mg/ml in DMSO.

      1. Protect from light.

      2. Store aliquots at -20 °C. Can be thawed and re-frozen.

    8. Pronase working solution.

      Dissolve 10 mg/ml of Pronase in water.

      Make fresh before each use.

    9. MitoTracker stock solution.

      Dissolve 0.53152 g/ml of MitoTracker in DMSO (1 mM).

      1. Protect from light.

      2. Store in small aliquots at -20 °C. Minimize freeze-thaw cycles.

    10. JC-1 stock solution

      Dissolve 1 mg/ml of JC-1 in DMSO (1.5 mM).

      1. Protect from light.

      2. Store in small aliquots at -20 °C. Minimize freeze-thaw cycles.

    Acknowledgments

    This work was supported by the NIH AG058610 and AG063947 grants to S.P.C. and AG000037 grat to A.M.H.

    This work has been adapted from Yen and Curran (2020), Yen et al. (2020), and Hammerquist and Curran (2020).

    Competing interests

    The authors declare no competing interests.

    References

    1. Altun, Z. F., Herndon, L. A., Wolkow, C. A., Crocker, C., Lints, R. and Hall, D. H. (Eds.). (2002-2021). WormAtlas.
    2. Amaral, A., Lourenco, B., Marques, M., and Ramalho-Santos, J. (2013). Mitochondria functionality and sperm quality. Reproduction 146(5): R163-174.
    3. Bonhoure, N., Byrnes, A., Moir, R. D., Hodroj, W., Preitner, F., Praz, V., Marcelin, G., Chua, S. C. Jr., Martinez-Lopez, N., Singh, R., Moullan, N., Auwerx, J., Willemin, G., Shah, H., Hartil, K., Vaitheesvaran, B., Kurland, I., Hernandez N. and Willism I. M. (2015). Loss of the RNA polymerase III repressor MAF1 confers obesity resistance. Genes Dev 29(9): 934-947.
    4. Grad, L. I. and Lemire, B. D. (2004). Mitochondrial complex I mutations in Caenorhabditis elegans produce cytochrome c oxidase deficiency, oxidative stress and vitamin-responsive lactic acidosis. Hum Mol Genet 13(3): 303-314.
    5. Hammerquist, A. M. and Curran, S. P. (2020). Roles for the RNA polymerase III regulator MAFR-1 in regulating sperm quality in Caenorhabditis elegans. Sci Rep 10(1): 19367.
    6. Huynh, T., R. Mollard and Trounson, A. (2002). Selected genetic factors associated with male infertility. Hum Reprod Update 8(2): 183-98.
    7. Jonassen, T., Marbois, B. N., Faull, K. F., Clarke, C. F. andLarsen, P. L. (2002). Development and fertility in Caenorhabditis elegans clk-1 mutants depend upon transport of dietary coenzyme Q8 to mitochondria. J Biol Chem 277(47): 45020-45027.
    8. LaMunyon, C. W. and Ward, S. (1998). Larger sperm outcompete smaller sperm in the nematode Caenorhabditis elegans. Proc Biol Sci 265(1409): 1997-2002.
    9. Oliva, A., Spira, A. andMultigner, L. (2001). Contribution of environmental factors to the risk of male infertility. Hum Reprod 16(8): 1768-1776.
    10. Practice Committee of the American Society for Reproductive Medicine. (2006). Effectiveness and treatment for unexplained infertility. Fertil Steril 86(5 Suppl 1): S111-4.
    11. Shakes, D. C. and Ward, S. (1989). Initiation of spermiogenesis in C. elegans: a pharmacological and genetic analysis. Dev Biol 134(1): 189-200.
    12. Sivandzade, F., Bhalerao, A. and Cucullo, L. (2019). Analysis of the mitochondrial membrane potential using the cationic JC-1 dye as a sensitive fluorescent probe. Bio-protocol 9(1): e3128.
    13. Ward, S., Hogan, E. and Nelson, G. A. (1983). The initiation of spermiogenesis in the nematode Caenorhabditis elegans. Dev Biol 98(1): 70-79.
    14. World Health Organization. (1987). Infections, pregnancies, and infertility: perspectives on prevention. Fertil Steril 47(6): 964-968.
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    16. Yen, C. A. and Curran, S. P. (2021). Incomplete proline catabolism drives premature sperm aging. Aging Cell 20(2): e13308.
    17. Yen, C. A., Ruter, D. L., Turner, C. D., Pang, S. and Curran, S. P. (2020). Loss of flavin adenine dinucleotide (FAD) impairs sperm function and male reproductive advantage in C. elegans. Elife 9: e52899.

简介

[摘要]不孕症是一个普遍存在且常常无法解释的问题。利用学习再现线虫男性报价小号洞察对人类男性的生育能力的个体因素的影响。我们创建了一组协议,以评估秀丽隐杆线虫精子质量的几个方面,包括数量,大小,激活率和线粒体形态。在诸如秀丽隐杆线虫这样的模型系统中研究精子生物学,可以访问为该生物体优化的丰富资源和技术,同时提供可能适用于其他系统的有价值的生物学信息。

图形摘要:

描绘秀丽隐杆线虫雄性的制备和后续精子质量测定的流程图


[背景]大约夫妻8%来自世界各地的不孕不育患(世界卫生组织,1987年),以及这些案件中30%有联合国已知的原因(在美国生殖,2006年的实践委员会).The多种因素影响男性生育,遗传和环境(奥利瓦等人。,2001;黄长发等人。,2002) ,在化妆研究人类容易混淆的影响。模型系统可用于调查单一因素对生育各个方面的影响。秀丽隐杆线虫是可遗传处理的生物,具有确定数量的细胞和生殖细胞,可以通过表型检查。

对哺乳动物线粒体功能(包括线粒体形态,线粒体基因组和拷贝数,线粒体蛋白质水平以及电子传输链复合物的酶活性)的研究表明,线粒体健康与精子功能之间存在联系(Amaral等,2013 )。此外,尽管这些途径的确切作用机理尚不清楚(Jonassen等人,2002 ;Grad和Lemire ,2004 ),但一些线粒体突变体对秀丽隐杆线虫的繁殖能力产生了负面影响。最近的工作从我们的实验室已证明MAFR-1 ,RNA的调节聚合酶III (Hammerquist和柯伦,2020 ),和ALH-6和PRDH-1 ,在线粒体脯氨酸的酶的分解代谢途径(日元等人,2020 ; Yen和Curran ,2021 )是秀丽隐杆线虫的正常精子功能所必需的,并且这些作用在哺乳动物模型中可能是保守的(Bonhoure等人,2015 ;Yen和Curran ,2021 )。

我们最近开发并发布了评估秀丽隐杆线虫精子质量的各种指标的方法(Hammerquist和Curran,2020;Yen和Curran,2020;Yen等人,2020)。一些我们提出,尽管添加了细节和精致工艺的方法,基本上是不变的,从原来的出版几十年前(沃德等人,1983;奶昔和Ward,1989; LaMunyon和Ward,1998) ,而另一些,特别是在精子线粒体融合的定量研究首先是在我们的实验室中进行的(Yen等,2020)。在这里,我们提供了一套全面检测的详细协议,是衡量质量和线粒体健康的精子。

关键字:秀丽隐杆线虫, 生育力, 精子质量, 精子形成, 线粒体



材料和试剂


显微镜载玻片(VWR,目录号:48312-004)
无菌6厘米P ETRI培养皿(VWR,目录号:25373-085)
铂金线(Tritech Research,目录号:PT-9010)
睫毛刷(由巴斯德移液器上的普通人睫毛组成,带胶带,通用)
显微镜载玻片盖玻片(VWR,目录号:48366-227)
PAP笔(液体阻断剂,目录号:Z377821)
可以密封的塑料或玻璃容器(Glasslock ,目录号:OCRT-048)
纸巾(通用)
封口膜(Sigma-Aldrich,目录号:P7543)
25G针(BD,目录号:305125)或手术刀(Technocut ,目录号:6008T-10)
750 ml离心瓶,可通过高压灭菌(通用)进行灭菌
2-200微升micropi p ETTE提示(杰纳西科学,目录号:24-151RL)
大肠杆菌OP50-1(可从秀丽隐杆线虫遗传学中心获得)
蠕虫病毒株(可从秀丽隐杆线虫遗传学中心获得)
氯化钠(Fisher Scientific,目录号:02-004-047)
蛋白ept (BD,货号:211820)
Bacto Agar(BD,货号:214040)
胆固醇(Sigma- Aldrich,目录号:C8667)
乙醇(VWR,目录号:89125-172)
氯化钙(Sigma- Aldrich,目录号:C3881)
硫酸镁(Sigma- Aldrich,目录号:M2773)
磷酸氢二钾(Sigma- Aldrich,目录号:P5504)
一元磷酸钾(Sigma- Aldrich,目录号:P0662)
硫酸链霉素(Sigma- Aldrich,目录号:S6501)
LB粉(Teknova ,目录号:L9315)
HEPES(j 。Ť 。贝克,目录号:4018-04)
氯化钾(Sigma- Aldrich,目录号:P3911)
BSA(Sigma - Aldrich,目录号:A9647)
右旋糖(j 。Ť 。贝克,目录号:JT1919)
磷酸氢二钠(Sigma- Aldrich,目录号:S9763)
氯化钾(Sigma- Aldrich,目录号:P3911)
海卫一X-100(Sigma- Aldrich,目录号:X100-100 m l )
异丙醇(BDH,目录号:BDH1133)
DAPI(Sigma- Aldrich,目录号:D9542)
凡士林(凡士林)
指甲油(透明,通用)
显微镜浸油(Fisher Scientific,目录号:12-624-66B)
Vectashield安装介质(VWR,目录号:H-1000)
Pronase (Millipore Sigma,目录号:53702)
MitoTracker TM Red CMXRos (Thermo Fisher,目录号:M7512)
MitoProbe TM JC-1检测试剂盒(赛默飞世尔(Thermo Fisher),目录号:M34152)
线虫生长培养基(NGM)板(请参阅食谱)
磷酸盐缓冲盐水(PBS)(请参阅食谱)
PBS + 0.01%Triton X-100(PBST)(请参阅食谱)
SM缓冲液(葡萄糖)(请参阅食谱)
SM缓冲区(BSA)(请参阅食谱)
M9 (请参阅食谱)
DAPI库存解决方案(请参阅食谱)
Pronase工作解决方案(请参阅食谱)
MitoTracker库存解决方案(请参阅食谱)
JC-1库存解决方案(请参阅食谱)
设备


2-20 μ升微量(吉尔森FA10003M)
20-200 μ升微量(吉尔森FA10005M)
规模(通用)
pH计(通用)
微量离心机,用于1.5 m l的试管(Eppendorf ,型号:5430)
带有用于750 m l离心瓶的摆桶式转子的冷冻离心机(贝克曼库尔特(Beckman Coulter),型号:Allegra X-15R)
管旋转器(Thermo Scientific ,目录号:88881001)
本生灯(通用)
解剖显微镜(Nikon SMZ 800,带光纤透照照明支架)
带有DIC,DAPI,GFP和RFP滤镜,10 × ,40-63 ×和100 ×物镜的复合显微镜(Zeiss ,型号:AxioScope5)
彩色相机(Zeiss AxioCam MRm )
蠕虫培养箱(通用,保持在20 °C)
软件


影像软件
取决于所用的显微镜(在这种情况下,我们使用Zeiss AxioScope和相关的ZEN成像软件)


ImageJ (可从NIH获得)
数据分析软件
我们使用GraphPad棱镜,尽管任何能够进行t检验的软件都足够。如果比较多个条件但并非绝对必要,方差分析可能会有所帮助。


程序


准备幻灯片(用于D.精子大小测定;E。精子激活测定;F。定性线粒体形态测定;以及G.定量线粒体融合测定)。
1.用一支PAP笔在显微镜载玻片上画一个直径为1-1.5 c m的圆圈。      注意:一个标准滑轨将很容易安装两个油脂圈,每个圈都有空间单独覆盖(使用22 × 22 mm正方形滑盖时)。


2.干燥过夜(或在层流通风橱中> 2小时)。      准备男性
1.保持在男性中生长的所需菌株的检测。      注意小号:


精子数量测定需要30至40名男性;每个重复的精子大小,激活和线粒体检测至少需要五只雄性。这个数字很容易从雄性和雌雄同体的混合人群中获得。为了确保所述在雄性繁殖的菌株使用,转让年轻成年男性和雌雄同体(3:1倍的比例),以新鲜平板小号4-5天采摘男性为之前的测定。
如果配合能力和/或雄性能育性受损,男性应当配合到雌雄同体(3:1倍的比例)上过夜NGM板用20接种μ升的25 × OP50-1(见配方1I说明用于进一步细节),并转移到库存NGM板。较小的食物点小号增加男性和雌雄同体之间接触的频率,因此增加了成功配合的可能性。伴侣动物之前采摘男性4-5天的试验。
如果较大的动物的数量是期望的,群可以通过碱性次氯酸盐处理,男性和雌雄同体的混合群体的同步描述之前(日元和柯伦,2020) 。为确保您有足够的男性,请只使用大约50%的男性人口。
2.挑选幼虫阶段4(L4)处女雄性股票NGM板小号自由雌雄同体对前一天测定(咨询WormAtlas (阿尔等人。,2021)用于进一步的信息,识别L4男性)。      注意小号:


挑选L4雄性可确保在测定之前不会通过交配而过早激活。男性应该以这种方式为所有测定法来制备,除了定性线粒体形态,为此,所述的MitoTracker染料应该转移雄性之前施加到食品的板小号(见步骤F)。
如果隔离时间过长,雄性往往会从板上爬下来寻找雌雄同体。如果这成为一个问题,能够通过允许雌雄同体(优选妊娠,未配合的)来抓取上接种到NGM嗅出防止男性的一些损失它与信息素和除去它们转移雄性板之前。
3.在20°C下孵育18-24小时。      精子ñ棕土试验
1.洗净雄虫从在用PBS + 0.01%的Triton X-100(PBST)隔离板一个1.5米升管。      2.以560 × g离心1分钟。除去上清液至0.1μm升(上使用标记的管作为一个指南)。      3.再用PBST清洗1-2次。      注意:这些洗涤物可从要染色的蠕虫中去除大肠杆菌。洗涤直至去除的上清液是透明的(并且不会被细菌混浊)。请勿超出此范围洗涤。其他洗涤增加丢失蠕虫而误吸的可能的上清液。


4.以560 × g离心1分钟。除去上清液至0.1 ml 。      5.添加600 μ升的40%的异丙醇和转动,以便在室温下3分钟以固定蠕虫。      6.虽然蠕虫固定,使染色搜索解决方案N:稀1毫克/米升DAPI原液至10微克/米升在40%异丙醇(10 μ升每1m DAPI升40%异丙醇)。      7.以560 × g离心1分钟。除去上清液至0.1 ml 。      8.添加600 μ升的染色在黑暗溶液并孵育5分钟。      9.以560 × g离心1分钟。除去上清液至0.1 ml 。      10.添加600 μ升PBST孵育在黑暗中进行30分钟至脱色。  11.在显微镜载玻片上安装样品小号(常规滑动    ,未那些在制备过程A)与的Vectashield封固剂。


12.盖上一个盖滑动和密封的用指甲油边缘。干燥(在黑暗中)5-10分钟。  13.带有DIC和DAPI过滤器的40-63 ×图像,确保在每个蠕虫中都可见所有精子。  注:精子细胞应限制在在雄虫精囊,可以从其他生殖细胞通过其紧凑的核区分开来。


14.收集每个精囊的z堆栈,确保捕获所有精子(如果需要缩小,包括所有精子,则使用40倍)。A R具有代表性图像是示于图1。  注意:在精囊的上边缘和下边缘(在精囊的每一侧都可以看到第一个精子)上设置边界,并在0.25微米的z平面距离上成像。该距离由Zeiss成像软件的自动设置确定。


图1.精囊中DAPI染色的精子。蠕虫的前部区域位于左侧,而后部则位于右侧。精囊呈黄色轮廓。比例尺,20μm 。


精子大小测定
1.清洁蠕虫以清除大肠杆菌。      可以通过以下两种方法之一进行清洁。对于第一种方法,移液管15〜两滴μ升M9到一个皮氏培养皿盖和使用一个铂丝蠕虫挑转移5天一个成年男性处女至M9的下降。使用的睫毛刷,转移蠕虫在到所述第二M9移动之前降他们到SM缓冲液上的准备滑动。对于第二种方法,使用一个铂挑传送>5天一个成年男性处女到一个未接种的NGM板和允许他们从爬行的食物点。Ť母鸡用干净的铂拾取(没有细菌)从转移5名男性的未接种的板的SM缓冲液上的制备的载玻片。在每个传输步骤中,分别小心地移动蠕虫。在没有食物的情况下拾起蠕虫需要练习,并且要以铲起的动作完成,此时镐头要伸到蠕虫的下方。


2.吸取35 μ升的SM缓冲液含有d extrose到所述上圆润滑脂的制备玻片(来自步骤A)。      3.转移5干净处女雄性的SM缓冲液上的使用任一滑动的睫毛刷或一个铂拾取,这取决于如何将动物进行清洗(见步骤D1)。      4.在一解剖显微镜,解剖蠕虫释放的精子。      使用手术刀或两个25号针头(横跨相互模仿针滑动的平坦表面剪刀刀片),切片之间男性的中点和尾部(从大约1/3体长的尾部)。
注意:快速移动,因为解剖的精子可能对蒸发引起的盐浓度和/或pH值变化敏感。


消毒的手术刀或针小号通过在≥70%的乙醇中浸渍和穿过样品之间一个本生灯火焰。使用前请先冷却。
5.盖解剖精子与一个盖玻片。      6.用指甲油密封。      7.图像的一个100 ×物镜使用一个DIC滤波器。A R具有代表性图像被显示于图2。      尽可能快地完成成像可能因为自发活化已经分离时精子被留下长时间观察到。在经验丰富的手中,此过程不应超过5分钟。
得分只有球形细胞和至少100每个重复。
注:通常情况下,100个精子细胞很容易从五个解剖每天一个滑,以获得一个成年男性。这可以通过对大约5-10个场成像来实现。字段的数量成像取决于如何在解剖利差他们的幻灯片。在成像时对精子的数量进行粗略的核算可能有帮助; 总是拍一些额外的图像。


图2.用于尺寸分析的分离精子的DIC图像。ImageJ中已概述了一个精子(黄色),用于大小分析。比例尺,10μm 。


精子活化测定
1.准备一个潮湿的房间(图3)      将湿纸巾放在有盖容器的底部。盖用石蜡膜的片材以创建一个干燥的表面上的地方滑动。
盖用的盖子。
图3 。DIY潮湿箱处于打开(A)和关闭(B)配置s


2.准备的链霉蛋白酶的工作溶液(见配方)和SM含有BSA和400毫微克/ μ升的链霉蛋白酶。      后的链霉蛋白酶工作溶液Ç ompletely溶解,稀4微升在96 μ升的SM缓冲液含有BSA。


3.清洁蠕虫除去大肠杆菌(参见P rocedure d详细小号)。      4.吸取35 μ升的SM缓冲液含有BSA到所述上圆润滑脂的制备玻片(来自步骤A)。      注意:使用含有BSA而不是葡萄糖的SM缓冲液进行激活测定。我们已经对两种补品进行了测试,并指出激活需要BSA。


5.转移5干净处女雄性的SM缓冲液上的使用任一滑动的睫毛刷或一个铂拾取,这取决于动物如何清洁(见步骤D1A)。      6.根据一个解剖显微镜,解剖蠕虫释放的精子(见步骤d的说明)。      7.解剖后的最终男性,添加35 μ升的SM缓冲液含有BSA和400毫微克/ μ升链霉蛋白酶。      8.孵育在所述湿室(有盖)15分钟。      注意:在我们的手中,15分钟可以激活大约80%的WT精子,但是激活对多种因素敏感,包括处理时间,温度和缓冲液成分的新鲜度。孵育时间可能需要根据个人经验确定。


9.在incubati上,制备的盖玻片。      使用镊子或一个移液管尖端,创建一个细线石油的果冻周围的边缘的盖玻片。
注意:我们公顷已经找到该密封的指甲油幻灯片影响小号活化并且因此仅使用凡士林密封载玻片用于该测定。


10.分钟后15,覆盖的解剖动物的准备盖玻片。不要进一步密封。  11.图像的一个100 ×物镜使用一个DIC滤波器。A R具有代表性图像被显示于图4。  注意:


由于精子可能位于不同的焦平面中,并且可能难以在单个图像中看到被激活的精子的假足,因此我们建议在两个不同的焦平面中对每个视场成像以帮助评分(此距离无需精确测量)。所述第二图像作为在确定是在第一不清楚精子细胞的活化状态的辅助一个。通常情况下,在拍摄之间的时间伪足的运动的图像也有助于揭示细节,可能无法在单个图像明显。
因为该盖片粘附到所述油浸渍后仅使用表面张力,改变目标幻灯片可能导致其移位或完全脱落。出于这个原因,检查了使用滑动一个低倍率(非油浸)成像之前的目标。浸入浸油后,请勿返回低功率物镜。
确保在孵育完成后的前5分钟内完成所有成像(添加SM / Pronase溶液后20分钟)。
每个重复至少得分100个精子细胞。
图4.在各种激活状态下,用于激活测定的分离出的精子的DIC图像,显示出尖峰(黑色箭头),叶状(黑色箭头)和完整的伪足(白色箭头)投影。请参考到图2为不活动的(圆形)精子细胞。比例尺,10μm 。


定性线粒体形态测定
1.将MitoTracker储备溶液(在DMSO中为1 mM)稀释到在M9中为100μM 。      2.添加50 μ升工作的MitoTracker溶液到所述NGM板50接种的食物点μ升的25 ×浓缩OP50和让干燥。      3.转移L4男性到所述准备好的板(从该使用任一之前的步骤)的睫毛刷或一个铂拾取,这取决于动物如何清洁(见步骤D1)。      4.在20 °C下孵育18-24小时。      5.清洁蠕虫除去大肠杆菌(参见步骤d为详细小号)。      6.吸取35 μ升的SM缓冲液含有葡萄糖到所述上圆润滑脂的制备玻片(来自步骤A)。      7.根据一个解剖显微镜,解剖蠕虫释放的精子(见步骤d的说明)。      8.盖的解剖精子细胞用一个盖滑动。      9.用指甲油密封。      10.图像的一个100 ×客观使用DIC和AlexaFluor594滤波器小号(RFP通道也应该工作)。代表图像是示于图5。  确保成像在10分钟内完成。
仅对球形细胞评分。
每次重复至少得分100个精子细胞。
图5. DIC(A)和AF594(B)通道中的MitoTracker染色精子。比例尺,2μm 。


定量线粒体融合测定
1.准备一潮湿室(见方法E详细小号)。        2.清洁蠕虫除去大肠杆菌(参见步骤d详细小号)。        3.准备染色液。        添加8 μ升1.5毫JC-1库存792 μ升的含有BSA(15 SM μM终浓度)。
4.吸取35 μ升的SM缓冲液含有BSA与JC-1到在上圆润滑脂的制备玻片(来自步骤A)。        5.转移5只清洗处女雄性的上SM缓冲液的使用任一滑动的睫毛刷或一个铂拾取,这取决于动物如何清洁(见步骤D1A)。        6.根据一个解剖显微镜,解剖蠕虫释放的精子(见步骤d的说明)。        7.解剖后的最终男性,添加25 μ升的SM缓冲液含有BSA和JC-1。        8.孵育在所述湿室(有盖)10分钟。        9.洗涤三次用100 μ升的含有BSA SM缓冲液(无JC-1)。        要小心,以避免完全祛瘀荷兰国际集团的从解剖精子的液体,因为这会影响精子细胞生理学(因此线粒体的表型)。
要小心,以避免溢出荷兰国际集团的油脂圈和losi纳克的样品。如果有必要,使用较小的冲洗体积(80-90 μ升)。
10.盖的解剖动物一个盖玻片并密封用指甲油。    11.图像与一个100 ×物镜使用AlexaFluor488(绿色)和AlexaFluor546(红色)通道。代表图像是示于图6。    注意:JC-1是线粒体特异性染料,可反映膜电位的变化。红色荧光(高膜电位线粒体物种)与绿色荧光(低膜电位线粒体物种)的比率是线粒体健康的常用指标(Sivandzade et al。,2019 )。


收集要记分的精子的Z堆栈。
注意:收集8-9图像与0.8微米在z平面上的间距。


确保成像是在10分钟内完成,如精子细胞是一个活(不固定),并可以经受线粒体的变化。
仅对球形细胞评分。
每个重复至少得分40个精子细胞。
图6.来自野生型(AB)和alh-6突变型(CD)动物的JC-1染色的精子细胞,分别显示出主要的球形和长方形线粒体。(B和D)ImageJ检测JC-1染色的精子线粒体。小号permatid大纲都标有白色虚线。比例尺,1μm 。该图像先前已发表在Yen等人的文章中。(2020 )。


数据分析


精子数量测定
1.反卷积z-stack。      2.计算z堆栈s中的精子细胞核。      注意:为了避免计算同一核不止一次或失去磁道的的一个小号计数,由画线和在一个时间计数一个区段划分精囊成多个部分。作为参考,在一个精囊的40倍图像堆栈中,我们选择了50-80个切片。部的数量将取决于蜗杆如何布置和安装介质是多少之间的盖滑动和滑动(在更小的体积,所述蠕虫可以是平坦的,并且较少的z栈将需要获取的图像,而可能需要更多的计数部分)。


3.每个复制品应至少包含10只动物。        4.至少应完成三个独立的重复实验。统计一nalysis应进行使用该学生的牛逼-测试。        注意:数据应表示为每只动物的平均精子数量(三个或三个以上重复样本中30个以上个体计数的平均值)。


尺寸测定
1.仅对聚焦的未激活的圆形精子评分。        2.每个重复样本应至少包含100个可用的精子细胞。        3.使用ImageJ的椭圆工具,按住Shift确保圆和轮廓,并测量符合精子的区域的纳入标准。        4.至少应完成三个独立的重复实验。统计一nalysis应使用执行该学生的牛逼-测试。        注意:数据应该被呈现为平均尺寸精细胞(的300+精子细胞个体在三个或更多个单独的重复的平均值)。


活化测定
1.使用每个视场的两个焦平面图像,对单个精子细胞进行激活(使用伪足)或未激活(没有伪足,包括正在被尖峰或波状投影激活的细胞)进行评分。      2.每个复制品应至少包含100个计分的精子细胞。      3.对于每个条件,计算所述百分比年龄的激活精子细胞(下总打进)。      4.至少应完成三个独立的重复实验。      5.计算平均激活率每重复和比较的样本使用的学生牛逼-测试。      注意:数据应表示为平均激活百分比(三个或三个以上百分比值的平均值,每个重复一个)。


定性线粒体形态测定
这是定性分析。


定量线粒体融合测定
1.采取分数的z栈与所述AlexaFluor546滤波器。使用Z堆叠小号来确定和得分线粒体是否是球形(未融合)或椭圆形的(稠合)。      2.每个复制品应至少包含40个单个精子细胞。      3.对于每个条件,计算的百分比的年龄稠合(下总的得分)线粒体。      4.至少应完成三个独立的重复实验。      5.使用比较数据的学生牛逼-测试。      注意小号:


数据应表示为每个精子融合线粒体的平均百分比(三个或三个以上重复样本的平均值为90+百分比)。
Ç膜电位hanges可以使用确定的红到利用绿色荧光通道比率的JC-1染料。ImageJ可用于荧光分析。
菜谱


线虫生长培养基(NGM)板
制备的1M硫酸镁的储备溶液4 ,5毫克/米升胆固醇,1M KH 2 PO 4 ,1中号的CaCl 2 ,和2.5%(W / V)链霉素。
溶解120.366克的硫酸镁4每1L水(1 M)。过滤消毒。
溶解5克的每1 L 100%乙醇(5毫克/米胆固醇升)。储存在4 °C 。
溶解136.086克的KH 2 PO 4每1L水(1 M)。过滤消毒。
溶解110.98克的氯化钙2每1L水(1 M)。过滤消毒。
每1 L的水(2.5%)溶解25 g的链霉素硫酸盐。过滤消毒。储存在4 °C 。
添加3克的氯化钠,17克的琼脂,将2.5g的蛋白胨,和950米升的水到玻璃烧瓶中。包括一个搅拌棒。
盖上箔纸并用高压灭菌器灭菌并溶解琼脂。
凉,有搅拌,55-60 ℃下。
加入1米升的1M硫酸镁的4 ,1米升5毫克/米的升胆固醇,25米升的1M KH的2 PO 4 ,1米升的1M的CaCl 2 ,和7.5米升的2.5%链霉素。
搅拌5分钟。
分配11米升成6厘米的Petri使用无菌技术的菜肴。
使其固化过夜。
种子储备NGM板用250 μ升的OP50-1过夜培养物(在LB中生长+链霉素,无晃动)。
注意:的MitoTracker染色(定性线粒体形态测定)需要25 ×浓缩的细菌。为了使这个,接种OP50-1在500ml的LB + streptomyci ñ。摇动生长,在37 °C过夜。离心20分钟5 ,000 ×克在4 ℃下(根据需要重复,以沉淀从整个培养细菌)。重悬于无菌M9中。将体积增加到20 ml 。小号撕扯4 ℃下进行长达6个月。


使用前让板干燥(覆盖)2-3天。
磷酸盐缓冲盐水(PBS)
添加26.5克的的Na 2 HPO 4 ·7H 2 O,80克的氯化钠,将2克的氯化钾,和2克KH 2 PO 4到一个玻璃瓶中。
将体积调至1L。
通过autoclav消毒ING 。
PBS + 0.01%Triton X-100(PBST)
使用切割移液管尖端(扩大开口)中,添加100 μ升的曲拉通X-100至1L无菌PBS。拌匀(不要摇晃)。


SM缓冲液(葡萄糖)
准备1 M HEPES,1 M NaCl ,1 M KCl ,1 M CaCl 2,1 M MgSO 4和1 M葡萄糖的储备溶液。
溶解238.3克的每1L水(1 M)HEPES。过滤消毒。
虽然在这一点不是必须的,该调节使pH值之前7.8溶液到的最终体积并灭菌将加快步骤4c中。


溶解58.4克的每1L水(1 M)的NaCl。过滤器或高压来消毒。
溶解74.6克的氯化钾每1L水(1 M)。过滤器或高压来消毒。
溶解110.98克的氯化钙2每1L水(1 M)。过滤器或高压来消毒。
溶解120.366克的硫酸镁4每1L水(1 M)。过滤器或高压来消毒。
溶解180.2克的每1L水(1 M)右旋糖。过滤器或高压来消毒。
结合2.5米升1M的HEPES,2.5M的升1M NaCl的,1.25微米的升的1M的氯化钾,250 μ升的1M的CaCl 2 ,50 μ升的1M硫酸镁的4 ,500 μ升的1M葡萄糖的和20μm左右升的水。
调节pH至7.8。
将体积增加到50 ml 。˚F ILTER灭菌。
储存在4 °C 。允许等分来给使用前RT。
SM缓冲区(BSA)
准备1 M HEPES,1 M NaCl ,1 M KCl ,1 M CaCl 2,1 M MgSO 4和5%(w / v)BSA的储备溶液。
溶解238.3克的每1L水(1 M)HEPES。过滤消毒。
尽管不是必需的,调节pH至7.8使前溶液吨Ó最终体积并灭菌将加快步骤5c中。


溶解58.4克的每1L水(1 M)的NaCl。过滤器或高压来消毒。
溶解74.6克的氯化钾每1L水(1 M)。过滤器或高压来消毒。
溶解110.98克的氯化钙2每1L水(1 M)。过滤器或高压来消毒。
溶解120.366克的硫酸镁4每1L水(1 M)。过滤器或高压来消毒。
溶解2.5克的每50米BSA升水(5%W / V)。过滤器来消毒。储存在4 °C 。
结合2.5米升1M的HEPES,2.5M的升1M NaCl的,1.25微米的升的1M的氯化钾,250 μ升的1M的CaCl 2 ,50 μ升的1M硫酸镁的4 ,1米升的5%( W / v)BSA,和20μm左右升的水。
调节pH至7.8。
将体积增加到50 ml 。˚F ILTER灭菌。
储存在4 °C 。使用前,让等分试样进入RT。
M9
添加30克的KH 2 PO 4 ,60克的的Na 2 HPO 4 ,将50g的NaCl和120毫克的硫酸镁4到一个玻璃瓶中。将体积调至1L。
通过autoclav消毒ING 。
高压灭菌后,可能会发生钙盐的沉淀。重新溶解可能需要几天的时间。搅动会有所帮助。


DAPI库存解决方案
溶解1毫克/米升在DMSO中。


避光。
将等分试样储存在-20 °C下。可以解冻并重新冷冻。
Pronase工作解决方案。
将10 mg / ml的Pronase溶解在水中。


每次使用前都要使其新鲜。


MitoTracker库存解决方案。
将0.53152 g / ml的MitoTracker溶解在DMSO(1 mM)中。


避光。
分装在-20 °C下。最大限度地减少冷冻-解冻循环。
JC-1储备溶液
将1 mg / ml的JC-1溶解在DMSO(1.5 mM)中。


避光。
分装在-20 °C下。最大限度地减少冷冻-解冻循环。
致谢


这项工作是由美国国立卫生研究院AG058610和AG063947支持赠款,以SPC和AG000037 GRAT到AMH


这项工作改编自Yen和Curran(2020),Yen等人。(2020),以及Hammerquist和Curran(2020)。


利益争夺


作者宣称没有利益冲突。


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Copyright Hammerquist 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. Hammerquist, A. M., Yen, C. and Curran, S. P. (2021). Analysis of Caenorhabditis elegans Sperm Number, Size, Activation, and Mitochondrial Content. Bio-protocol 11(11): e4035. DOI: 10.21769/BioProtoc.4035.
  2. Yen, C. A., Ruter, D. L., Turner, C. D., Pang, S. and Curran, S. P. (2020). Loss of flavin adenine dinucleotide (FAD) impairs sperm function and male reproductive advantage in C. elegans. Elife 9: e52899.
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