Laboratory methods for the detection of chromosomal structural aberrations in human and mouse spermatozoa by fluorescence in situ hybridization

Summary

This new generation of sperm F I S H assays has identified multiple paternal risk factors such as age, polypharmacy, lifestyle, and environmental aspects and occupational exposures. These sperm F I S H assays provide new opportunities to identify and characterize male reproductive risks associated with genetic, lifestyle, and flavin factors. This chapter provides an overview of the laboratory methods used to detect human (A C M assay) and mouse (C T 8 Mao assay) spermatozoa with chromosomal structural aberrations that have been shown to be effective in detecting stem cell environmental mutagens.

By Martin, this experiment is from "Environmental Genomics Laboratory Guide".

Operation method

Laboratory methods for the detection of chromosomal structural aberrations in human and mouse spermatozoa by fluorescence in situ hybridization

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I. Materials and reagents 1 Preparation of semen smear

(1) 3 to 9 uL of fresh or frozen semen (see Note 1).

(2) Glass slide with frosted ends.

(3) 1 0 0 % ethanol.

2 De-concentration of spermatozoa

(1) Add 40 mL of dithiothreitol (D TT, Sigma-Aldrich, St. Louis, MO) to the staining vat and place on ice.

(2) 40 mL of lithium 3,5-diiodosalicylate (LIS, Sigma-Aldrich) was added to the staining vat and placed at room temperature (R T ).

(3) Tris (hydroxymethyl) aminomethane hydrochloride (Tris-H C l , Sigma); adjust p H to 7. 8.

(4) Autoclaved distilled water (d H20 ).

(5) Circulating water bath set to 77~78°C.

Continue to add water until the volume reaches 1()00 mL. Filter to remove bacteria and store at room temperature.

3 Laboratory methods for human ACM determination

3.1 Semen smear preparation

(1) Soak slides in 100% ethanol for at least 2 days. Layer the slides in a crisscross pattern in a covered jar or container to expose as many slides as possible to the alcohol. Fill the jar with alcohol until all slides are covered. When removing the slides, wear appropriate personal protective equipment (PPE), wipe all slides vigorously with a Kimwipe clean room wipe, and place them in the slide box.

(2) Using a diamond pen, cut a piece from another clean slide, slightly less than the width of the slide (use another slide to measure); after marking the surface, break the slide inward, carefully cracking the slide. Be careful not to touch the edge that will be in contact with the sample. Alternatively, a coverslip can be used instead; however, coverslips are more likely to break during the coating process. Wear appropriate personal protective equipment during the procedure.

(3) Pipette a 7uL semen sample onto a clean glass slide. The volume can be adjusted according to the sperm concentration of the sample (e.g., 3 to 9 uL) (see Note 2 for low sperm count donors).

(4) Use a pre-cut slide to coat the sample. Holding the frosted end in one hand to tilt the slide slightly and the other end stationary on the bench, place the cut slide (or coverslip) directly over the sample drop; the clean, untouched prefabricated edge is allowed to come into contact with the slide only at this point. Slowly move the edge of the cut slide coverslip into contact with the sample; the sample will automatically follow the edge. After the sample is distributed down the edge , drag the edge toward the frosted end and smear the sample 2. 5 to 4 c m .

(5) Place the coated slide flat on a clean paper towel. While the sample is drying, place the coated slides in a slide box with the lid slightly open and allow them to air-dry for at least 24 h . We strongly recommend leaving the slides for several days (see Note 3).

3.2 ACM probe preparation

This procedure uses random priming to generate labeled probes for hybridization. Random octamers are polymerized with the denatured DNA template and extended with Kleenow fragments. Procedure
During this procedure, fluorescently labeled 11- d U T P was discharged in 10- to 40-fold

4. Microscopic data collection program for sperm F I S H determination

Laboratory protocols for making good hybridization quality slides for the A C M and C T 8 assays have been described in previous sections. Hybridization quality, i.e., a highly bright and compact F I S H signal and low background, is the most critical factor influencing the success of sperm F I S H studies. However, visualization of the F I S H signal and data collection protocols developed to minimize the impact of technical factors on experimental results are equally important (see Pacchierotti and Sgura, this book, for information on fiducial light microscopy and filter settings). In what follows, we will focus on several technical factors that are critical to the reliability of the sperm FISH assay, such as (1) the development of rigorous recording criteria for determining whether a spermatozoon contains an abnormal number of spots; (2) the process of recorder blinding and data collection; and (3) the harmonization of recording criteria among different recorders (see Note 16).

4.1 Recording standards for ACM and CT8 sperm FISH assays

The subjective evaluation of FISH signals is a problem that can lead to large variations in recording results between different experimenters and laboratories. Our research group has invested considerable effort in developing rigorous recording standards to minimize inter-laborator variation. Rigorous enforcement of these recording standards is necessary to produce reproducible data. The first step in the recording criteria is to decide whether a sperm should be recorded. The sperm being recorded must meet the following conditions (these conditions apply to both human and mouse sperm):

(1) The entire cell must be visible. Only count cells where the entire cell edge is visible. Do not count cells that are partially obscured by other cells. For example, do not count cells in large clusters; overlapping cells may obscure the synaptic signal.

(2) Cells must appear to be intact. The contents of the cell must be retained within the cell margins. Sometimes overly de-concentrated cells move out of the nuclear capacitance or fluorescence region and are not suitable for recording. For example, do not count cells with fluorescent areas outside the body of the sperm head.

(3) Cells should be in areas that hybridize well on the slide. Usually on hybridization slides, there are entire areas that hybridize effectively as well as areas that do not hybridize well. It is necessary to ignore certain areas that do not hybridize well.

(4) The size of the cells should be not less than 5um and not more than 15u m . An eyepiece with an index line can be used for this purpose. Undeconcentrated cells do not hybridize reliably, and to avoid biased data, cells smaller than 5u m on the index line are not included in the denominator. Fractions of such cells are retained, but their fluorescent regions are not recorded. Overly de-concentrated cells exhibit a very diffuse hybridization signal that also cannot be reliably recorded.

(5) Cells should be free of background. There may be some background signal on the slide. An area with too much background makes its true signal difficult to distinguish and should not be recorded.

Only sperm that meet all the conditions should have their F I S H phenotype recorded. Below, we describe the decision tree for the A C M assay and the decision tree for the C T 8 assay.

4.2 Decision tree for the ACM assay

Before analyzing the F I S H signal, the recorder should use phase contrast microscopy to evaluate the outline of the cell (intact or overflow), the presence of one or more tails, to determine if the recorded cell is eligible. Similarly, it must be determined that it is one cell and not two overlapping cells. The following criteria must be met when recording:

(1) The entire cell is visible, with no portion hidden from view.

(2) The edge of the cell appears intact.

(3) Cells have a tail or tail attachment zone (see Note 17).

(4) All hybridization signals are within the cell.

(5) Cell size is not less than 5 and not more than 15 scales as measured on a graduated scale.

(6) Cells are on an area of the slide where most of the cells are well hybridized.

If any of the conditions are not met, the cells are not suitable for recording. If the contents are overflowing or the cells are too small or too large, this information should be counted, but the hybridization signal is not recorded. If the cell is suitable for recording, the steps in the decision tree below are used to determine the F I S H phenotype.

4.2.1 Is there a break between the Alpha(A ) and Classic (Q probes?).

To be defined as a break between A and C probes the following conditions must be met:

(1) The A and C signals must be separated by at least the width of the A signal region.

(2) Both signals must be normal in size on the same area of the slide, depending on the hybridization signal that these probes are intended to show.

(3) These signals must be completely separated and must not have any wires connected to them.

(4) The cells have a tail or tail attachment zone.

(5) There is no hybridization signal around or within the cell suggesting it may be artifactual.

If all conditions are met, the cell may be recorded as having a break between the A and C regions; otherwise the cell is not recorded.

4.2.2 Is there a break within the Classic (C ) probe?

To be defined as an interruption within the C signal, the following conditions must be met:

(1) Only one C signal is connected to the A signal.

(2) If the two C signals are of equal size, they must be separated by the width of a separate signal; if unequal in size, they must be separated by the width of the larger C signal.

(3) The two signals must have approximately the same strength.

(4) The signals must be completely separated, with no wires connected.

(5) Cells have tails or tail attachment zones.

(6) There is no hybridization signal around or within the cell suggesting it may be artifactual.

If all conditions are met, the cell may be recorded as having a break in the middle of the C region; otherwise the cell is not recorded.

4.2.3 Are any signals duplicated or deleted?

To be defined as a duplicate in the C area, the following conditions must be met:

(1) The two C signals in the cell must have approximately the same size.

(2) The two signals must be of approximately the same intensity.

(3) Both signals must be of normal size on the same area of the slide, depending on the hybridization signal that these probes are intended to show.

(4) The signals must be separated by at least one full area width.

(5) Signals must be completely separated with no wires connected.

(6) Cells have a tail or tail attachment zone.

(7) There is no hybridization signal around or within the cell suggesting that it may be artifactual.

If all conditions are met, the cell may be recorded as having a duplicate in the C region; otherwise the cell is not recorded.

To be defined as a deletion in the C region, the following conditions must be met:

(1) The deletion of the C signal must be identified with a filter that specifically targets such a signal.

(2) There is no debris near or around the cell.

(3) Cells are not damaged.

(4) Cells have tails or tail attachment zones.

If all conditions are met, the cell may be recorded as having a deletion in region C; otherwise the cell is not recorded. A similar procedure is used to determine if there are duplicates or deletions in the A and M regions.
To be defined as a duplication in the A C probe, the following conditions must be met:

(1) The two signals for each probe must be approximately the same size.

(2) The two signals from each probe must be approximately the same strength.

(3) Both signals of each probe must be of normal size on the same area of the slide, depending on the hybridization signals that these probes are intended to show.

(4) The signals must be separated by at least one full area width.

(5) Signals must be completely separated with no wires connected.

(6) Cells have a tail or tail attachment zone.

(7) There is no hybridization signal around or within the cell suggesting that it may be artifactual.

If all conditions are met, the cell may be recorded as having duplicates in regions A and C; otherwise the cell is not recorded.

To be defined as a deletion in the A C region, the following conditions must be met:

(1) The deletion of A and C signals must be determined using triple, single, and D A P I filters.

(2) There is no debris near or around the cell.

(3) Cells are not damaged.

(4) Cells have tails or tail attachment zones.

If all conditions are met, the cell may be recorded as having a deletion in the A C region; otherwise the cell is not recorded.

4.2.4 Are the cells diploid spermatozoa?

To be defined as a diploid spermatozoa, the following conditions must be met:

(1) There are two A signals, two C signals, and two M signals.

(2) The two signals of each probe must be approximately the same size.

(3) The two signals of each probe must be approximately the same strength.

(4) Both signals of each probe must be of normal size on the same area of the slide, depending on the hybridization signals that these probes are intended to show.

(5) The two signals of each probe must be separated by at least one full area width.

(6) The signals must be completely separated without any wires connected.

(7) The absence of hybridization signals around or within the cell suggests that it may be an artifact.

(8) The cell is indeed one cell and not two overlapping cells (this needs to be scrutinized with a phase contrast microscope).

If all conditions are met, the cell may be recorded as diploid; otherwise the cell is not recorded.
Occasionally, spermatozoa without hybridization signals may be seen. In these cases, the absence of signal should be confirmed with all filters. Additionally, the cells should be verified under a phase contrast microscope to be of the required size, with no debris overlapping the mirror and no cells overlapping. After the above criteria were passed, the cells were recorded as ◦ (Table 1).

4.3 Decision tree for CT8 determination

Prior to analyzing the F I S H signal, the recorder should use phase contrast microscopy to evaluate the outline of the cell (intact or overflow), the hook shape of the head, and the presence of one or more tails to determine if the recorded cell is eligible. Similarly.

must also be determined to be one cell rather than two overlapping cells. When recording, the following criteria must be met:

(1) The entire cell is visible, with no portion hidden from view.

(2) The edge of the cell appears to be intact.

(3) Cells have hooks.

(4) All hybridization signals are inside the cell.

(5) The cell is on the area of the slide where most of the cells hybridize well.

If any of the conditions are not met, the cells are not suitable for recording. If the contents are overflowing or the cells are too small or too large, this information should be counted, but the hybridization signal is not recorded. If the cell is suitable for recording, the steps in the decision tree below are used to determine the IrI S H phenotype.

4.3.1 Are there duplications or deletions in the mitotic regions of chromosome 2?

For a duplication to be defined as a C region, the following conditions must be met:

(1) The two C signals in the cell must have approximately the same size.

(2) Both signals must have approximately the same intensity.

(3) Both signals must be normal in size on the same area of the slide, depending on the hybridization signal that these probes are intended to show.

(4) The signals must be separated by at least one full area width.

(5) Signals must be completely separated with no wires connected.

(6) Cells have a hook shape.

(7) There is no hybridization signal around or within the cell suggesting it may be artifactual.

If all conditions are met, the cell may be recorded as having a duplicate in the C region; otherwise the cell is not recorded. To be defined as a deletion in the C region, the following conditions must be met:

(1) The deletion of the C signal must be identified with a filter that specifically targets such a signal.

(2) There is no debris near or around the cell.

(3) Cells are not damaged.

(4) Cells have hooks or tails.

If all conditions are met, the cell may be recorded as having a deletion in region C; otherwise the cell is not recorded.

4.3.2 Are there duplications or deletions in the telomeric region of chromosome 2?

For a duplication to be defined as a T region, the following conditions must be met:

4.2.5

1 Both T signals within a cell must have approximately the same size.

2 The two signals must have approximately the same intensity.

3 Both signals must be normal in size on the same area of the slide, depending on the hybridization signal that these probes are intended to show.

4 The signals must be separated by at least one full area width.

5Signals must be completely separated with no wires connected.

6 Cells have a hook shape.

7 There is no hybridization signal around or within the cell suggesting that it may be artifactual.

If all conditions are met, the cell may be recorded as having a duplicate in the T region; otherwise the cell is not recorded.

To be defined as a deletion in the T region, the following conditions must be met:

1 The deletion of a signal must be identified with a filter that specifically targets such a signal.

2 There is no debris near or around the cell.

3 Cells are not damaged.

4 Cells have hooks or tails.

If all conditions are met, the cell may be recorded as having a deletion in the T region; otherwise the cell is not recorded.

4.3.3 Is chromosome 2 of this cell aneuploid?

To be defined as a disomy of chromosome 2, the following conditions must be met:

1 The two intracellular C signals and the two T signals must be approximately the same size.

2 The intensity of the two signals for each probe must be approximately the same.

3 On the same area of the slide, both signals of each probe must be normal in size, depending on the hybridization signal that these probes are intended to show.

4 The two signals of each probe must be separated by at least one full area width.

5 The two signals of each probe must be completely separated without any wires connected.

6 Cells must be hook-shaped.

7 The absence of hybridization signals around or within the cell suggests that it may be artifactual.

If all conditions are met, the cell may be recorded as a disomy of chromosome 2; otherwise the cell is not recorded.

To be defined as a deletion of chromosome 2, the following conditions must be met:

The deletion of the 1C and T signals must be determined using filters specific to each signal.

2There is no debris near or around the cell.

3 Cells are not damaged.

4 Cells have hooks or tails. ,

If all conditions are met, the cell may be recorded as having a deletion of chromosome 2; otherwise the cell is not recorded.

4.3.4 Is chromosome 8 aneuploid in this cell?

To be defined as a disomy of chromosome 8, the following conditions must be met:

1 Both intracellular 8 signals must be approximately the same size.

2 Both signals must be approximately the same intensity.

3 The two signals must be normal in size on the same area of the slide, depending on the hybridization signal that these probes are intended to show.

4 The two signals must be separated by at least one full area width.

5The two signals must be completely separated without any wires connected.

6 The cells must be hook shaped.

7 The absence of hybridization signals around or within the cell suggests that it may be art

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