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Chapter 18 - Sperm Chromatin Structure: Toluidine Blue Staining

Published online by Cambridge University Press:  05 April 2021

Ashok Agarwal
The Cleveland Clinic Foundation, Cleveland, OH
Ralf Henkel
University of the Western Cape, South Africa
Ahmad Majzoub
Hamad Medical Corporation, Doha
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Gametogenesis is a central biological process for sexual reproduction. In this process, both haploid male and female gametes are needed for successful fertilization. Male gamete or sperm are produced during spermatogenesis; a complex, unique, and tightly regulated process, which includes a series of physiological, biochemical and morphological events. During this process, round diploid spermatogonia are differentiated into haploid spermatozoa with an acrosome, flagellum, and condensed nucleus. Condensation of nucleus or chromatin compaction play a paramount role in formation of morphology, especially the size of sperm. Therefore, during spermiogenesis, nucleo-histones are replaced by protamines. To achieve this aim, histones become hyper-acetylate to reduce their binding affinity to DNA. Subsequently, hyper-acetylated histones are replaced by transition proteins and next by protamines. According to the literature, in the human, 85–95 percent of the histones are replace with protamines while 5–15 percent of histones remain bounded to mature sperm DNA in humans [1, 2]. Therefore, through this process, sperm chromatin becomes six-fold more condensed than chromatin of other cells. This condensation will protect sperm chromatin from chemical and mechanical damages, bacterial infections, detrimental molecules such as oxidants, and also damaging molecules within the female reproductive system [2]. Alterations in the sperm histone/protamine ratio due to reduction of hyper-acetylation of histones or excessive histone retention are associated with abnormal chromatin packaging which can increase susceptibility to DNA damage, and eventually result in male infertility [1]. Therefore, normal chromatin packaging in the spermatogenesis process is essential for maintaining genomic integrity and accomplishment of fertility. In this regard, numerous studies have shown that there is a significant positive correlation between proper sperm chromatin condensation with clinical outcomes of infertile couples being candidates for assisted reproductive technology (ART) [3]. In addition, a significant positive association has been reported between sperm abnormal chromatin packaging with recurrent pregnancy loss [4]. Therefore, numerous studies suggested that the evaluation of "sperm chromatin condensation" may have a prognostic value in the assessment of male infertility. For this aim, several analytical methods based on cyto-chemical or fluorescent dyes have been proposed for the assessment of sperm nuclear compaction such as acidic aniline blue (for direct detection of excessive presence of histones), toluidine blue staining and chromomycin A3 (for indirect assessment of protamine deficiency), sperm chromatin structure assay (SCSA) or acridin orange test (for indirect assessment of sperm chromatin DNA stability based on chromatin compaction termed "high DNA stability structure"). Toluidine blue (TB) staining is one of the procedures used for evaluation of chromatin structure. Therefore, this chapter, provides detailed practical advice on TB staining, optimization, and interpretation of results in the field of medically assisted reproduction.

Publisher: Cambridge University Press
Print publication year: 2021

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