The integrity of mammalian sperm DNA is of prime importance for the paternal genetic contribution to normal offspring. Damaged DNA in the single sperm that fertilizes a female oocyte can have a dramatic negative impact on fetal development and health of the offspring throughout adult life.
Animal and human fertility clinics typically assess semen quality by measuring sperm density, total count, motility, and morphology. Clinics rarely measure sperm DNA integrity, perhaps because they are unaware of the availability of, or lack the instrumentation for, a rapid, reliable, and practical test. The sperm chromatin structure assay (SCSA; see Basic Protocol) is one such test whose importance is becoming increasingly recognized. Whereas light microscope measurements are commonly made on 100 to 200 sperm per sample, the SCSA can utilize a fresh (or frozen-thawed) semen sample and collect data on 5000 or more cells in just a few minutes. Figure 7.13.1 shows two examples of a SCSA clinical report that can be generated and sent to a clinician by FAX or e-mail within 15 to 30 min of measurement.
A protocol for SCSA is outlined in this unit (see Basic Protocol). Support Protocol 1 describes flow cytometer alignment and setup. Support Protocol 2 outlines sample collection and storage conditions so that all experimental data is related back to the standard; the need for a reference sample is highlighted. Support Protocol 3 details the very important analysis of SCSA data; means to generate the calculated parameter of a [at = red/(red + green)] both on and off line are discussed. Support Protocols 4 and 5 detail methods to obtain purified sperm nuclei that are needed for analysis of nuclear DNA (eliminating mitochondrial DNA) or nuclear -SH groups (eliminating the large number of -SH groups in the sperm tail). Sonication is sometimes used to verify that red fluorescence is derived from denatured DNA rather than residual cytoplasmic RNA. Rat sperm are always sonicated since the very large tails tend to plug the flow cell. Support Protocol 6 details fixation of sperm in ethanol.
SCSA data on thousands of semen samples from humans (Evenson 1997, 1999a; Evenson et al., 1991, 1999; Fossa et al., 1997; Larson et al., 1999, 2000; Grajewski et al., 2000), bulls (Ballachey et al., 1987, 1988; Evenson, 1999b), stallions (Evenson et al., 2000b), boars (Evenson et al., 1994), and exotic cats (unpublished) show the clinical value of this assay for human/animal fertility assessment.
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