Chromomycin A₃‐staining as an indicator of protamine deficiency and fertilization

Abstract

Mature mammalian spermatozoa have a compact and stable nuclear structure conferred by protamines instead of histones, which are present in all other cellular types. Chromomycin A3 (CMA3) is a useful tool for the detection of protamine deficiency in sperm chromatin. The purpose of this study was to correlate the percentage of spermatozoa staining positively for CMA3 with sperm parameters and in-vitro fertilization of human oocytes. Spermatozoa were collected from 56 fertile and 18 infertile men, and washed twice in PBS, fixed in two changes of methanol : acetic acid (3 : 1 v : v) spread on rinsed slides treated with APES and dried. Twenty-four of the semen samples were subjected to both Percoll and swim-up, and were stained subsequently with CMA3. CMA3-stained spermatozoa were expressed as a percentage in a count of 200 spermatozoa. A substantial variation in the percentage of CMA3-stained cells was observed in ejaculated human spermatozoa, varying between 8% and 77%. A strong negative correlation (r = -0.64, p < 0.001) was found between sperm count and the percentage of CMA3-stained spermatozoa. No correlation was found between CMA3-stained spermatozoa and their motility, while excessive sperm morphological abnormalities were related positively to CMA3-staining. Spermatozoa in samples exhibiting low (8-62%) CMA3-staining had significantly higher fertilizing rates in vitro than did samples exhibiting high (49-77%) CMA3-staining. The mean percentage of CMA3-stained spermatozoa after swim-up or Percoll preparation (26% vs 31%) did not differ significantly. These results demonstrate a close relationship between CMA3-staining, fertilization and sperm count, and suggest potential application of this marker for the prediction of sperm quality and fertilizing capacity.

Sperm Chromatin Structure Assay: Its Clinical Use for Detecting Sperm DNA Fragmentation in Male Infertility and Comparisons With Other Techniques

“…Neither Lolis et al (1996) nor found a significant change in CMA 3 staining following swim-up preparation, confirming the absence of a relationship between CMA 3 staining and motility. However, a significant decrease in CMA 3 staining was reported following density gradient centrifugation by , although not by Lolis et al (1996). The decrease in CMA 3 staining is somewhat intuitive due to the selection of highly condensed spermatozoa by density gradient preparations.…”

“…These results indicate that a high percentage of morphologically normal spermatozoa may have hidden abnormalities in chromatin structure. However, no clear cutoff value for infertility has been demonstrated (Lolis et al, 1996). While one study reported an association between CMA 3 staining and fertilization ability following SUZI, this was not found in routine IVF or ICSI .…”

“…CMA 3 staining is related to low sperm counts, abnormal morphology, and low in vitro fertilization rates Lolis et al, 1996;Esterhuizen et al, 2000;Iranpour et al, 2000). Neither Lolis et al (1996) nor found a significant change in CMA 3 staining following swim-up preparation, confirming the absence of a relationship between CMA 3 staining and motility.…”

Identification and Characterization of Alternatively Transcribed Form of Peroxiredoxin IV Gene That Is Specifically Expressed in Spermatids of Postpubertal Mouse Testis

“…Condensation of spermatid chromatin is accompanied by replacement of histones with protamines, which are basic nuclear proteins with a high Cys content, as well as by subsequent oxidation of protamine thiol groups to form disulfide linkages that result in protein crosslinking (32,34). The chromatin of sperm is highly compacted to protect the DNA from deleterious environments with insufficient oxidation of protamine thiol groups being associated with damage to sperm DNA (35)(36)(37). The reduction of sperm disulfides followed by decondensation of sperm chromatin in readiness for fertilization is accomplished in oocytes, which contain high levels of glutathione (38).…”

Changes in spermatozoal chromatin packaging and susceptibility to oxidative challenge during aging

“…The CMA3 assay is becoming more extensively used for evaluating sperm quality as part of male fertility assessment (52,53). It provides an indirect measure of assessing protamines in sperm (54,55), where CMA3 competes with protamines for chromatin binding (47,56).…”