Development of a thiol-reactive fluorescent probe for the identification of human spermatozoa
Adams, Shannon Michele
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Current methods of identifying semen include preliminary methods such as an alternate light source (ALS) and color tests which test for the presence of acid phosphatase (AP). Confirmatory methods for identifying semen include the microscopic identification of spermatozoa which typically use staining methods such as the Christmas Tree Stain (KPIC) or hematoxylin and eosin stain (H&E). Other methods include immunoassay cards that test for the components of semen such as prostate specific antigen (PSA) and semenogelin (Sg). One common fluorescent staining method used to identify the presence of spermatozoa is SPERM HY-LITERTM which uses an anti-human sperm-specific mouse monoclonal antibody coupled to an AlexaFluor 488 dye. This causes the entire head of the sperm cell to fluoresce when a FITC filter is used. It also utilizes a 4,6-diamidino-2- phenylindole (DAPI) fluorescent dye that stains all cell nuclei non-specifically. This allows the analyst to easily identify whether sperm is present in the sample. A drawback of the antibody labeling procedure is that there are many washing and transfer steps that can lead to sample loss; thus a need for a new, optimized staining method exists. During spermiogenesis, protamines replace histones to further condense the DNA of the sperm nucleus. Humans express two proamines, protamine 1 (P1) and protamine 2 (P2). The protamines contain an arginine-rich core as well as cysteine residues. The high levels of arginine create a net positive charge that allows for stronger binding to DNA. The cysteine residues allow for the formation of inter and intra-protamine disulfide bonds which allow for the chromatin compaction. If the disulfide bonds found in protamines can be reduced to produce free thiols, a thiol-reactive probe can bind and label the protamines in the sperm nucleus. Reduction of the disulfide bonds can be performed by use of reagents such as dithiothreitol (DTT) or tris-(2-carboxyethyl)phosphine (TCEP). From the literature on thiol-reactive probes, it is suggested that TCEP be used as a reducing agent due to its structure. DTT contains thiol groups and when removed from the sample, thiol groups may be oxidized back into disulfide bond. In addition, TCEP is more stable than DTT at a higher pH and higher temperatures. Once disulfide bonds have been reduced, a thiol-reactive probe may be introduced. There are many different types of probes that may be used. Maleimides are commonly used for thiol modification and quantitation. When the compound encounters a thiol group, the thiol group is added across the double bond, yielding a thioether. The reagent used is N-7-dimethylamino-4-methylcoumarin-3-yl)maleimide (DACM). It absorbs light at 376 nm and emits light at 476 nm, producing a blue fluorescence. The dye is nonfluorescent until it reacts with a thiol group. In this research, both sperm and epithelial cells were added to the slides in order to develop a novel staining procedure. The initial protocol used TCEP to break the disulfide bonds followed by DACM to bind free thiol groups. Sodium dodecyl sulfate (SDS) was then added to the protocol to lyse cells. DTT was tested for use as a reducing agent as well. The purpose of establishing this protocol was to design a procedure for rapid labeling of sperm that does not require antibody labeling. In the antibody labeling procedure, there are many washing and transfer steps. The proposed method may limit the number of procedural steps resulting in less loss of biological material. It can also help to limit the time-consuming methods of the current staining techniques, such as KPIC and H&E.