The paternal genome in many animal taxa is efficiently packaged into the sperm nucleus by switching from a histone (nucleosome)-based chromatin configuration to one using predominantly protamines. Nonetheless, various studies have shown that some nucleosomes, often containi ...

Epigenetic modifications on the DNA sequence (DNA methylation) or on chromatin-associated proteins (i.e., histones) comprise the “cellular epigenome”; together these modifications play an important role in the regulation of gene expression. Unlike the genome, the epigenome is hi ...

Protamines, sperm-specific nuclear proteins, are essential for sperm chromatin condensation and DNA stabilization. They are small, highly basic, and rich in disulfide bonds. Under reducing conditions, protamines, along with other basic proteins, are soluble in acid solutions. Be ...

The isolation of mammalian sperm heads from their tails is complicated by the relatively high density of the tails, but facilitated by the fact that protamine condensation of the sperm chromatin and the insolubility of the perinuclear theca make the sperm nucleus stable in sodium dodecyl sul ...

In this chapter, the laboratory methods for detection of sperm biomarkers that are aimed at identifying arrested sperm development are summarized. These probes include sperm staining with aniline blue for persistent histones, representing a break in the histone-transition prote ...

Proteomics is the study of the proteins of cells or tissues. Sperm proteomics aims at the identification of the proteins that compose the sperm cell and the study of their function. The recent developments in mass spectrometry (MS) have markedly increased the throughput for the identificati ...

Rapidly advancing tools for genetic analysis on a genome-wide scale have been instrumental in identifying the genetic bases for many complex diseases. About half of male infertility cases are of unknown etiology in spite of tremendous efforts to characterize the genetic basis for the diso ...

The isolation of spermatozoal RNA is a challenging procedure due to the intrinsic heterogeneous population of cells present in the ejaculate and the small quantity of RNA present in sperm. The transcriptome of these gametes includes a wide variety of messenger RNAs (mRNAs), small noncoding ...

High quality DNA extractions developed for mammalian somatic cells are ineffective for sperm, due mainly to the high degree of nuclear compaction in sperm. The highly specialized nuclear proteins in sperm create a chromatin structure that is at least six times denser than histone bound DNA. U ...

An improved enhanced chemiluminescence antioxidant assay utilizes horseradish peroxidase conjugate and luminol to produce a cell-free oxygen radical generating system. We introduce the use of a peroxidase enzyme stabilizer to prolong the production of oxygen radicals at a stea ...

The chemiluminescence method is the most commonly employed technique as a direct measurement of reactive oxygen species (ROS) generation by spermatozoa. This assay is capable of quantifying both intracellular and extracellular ROS. Moreover, the use of various probes allows for dif ...

Transmission electron microscopy (TEM) studies have provided the basis for an in-depth understanding of the cell biology and normal functioning of the testis and male gametes and have opened the way to characterize the functional role played by specific organelles in spermatogenesis a ...

Immunohistochemistry is an important technique that uses specific antibodies to determine the cellular localization of proteins/antigens in highly complex organs and tissues. While most immunohistochemistry experiments target protein epitopes, nonprotein antige ...

Male germ cells in all mammals are arranged within the seminiferous epithelium of the testicular tubules in a set of well-defined cell associations called stages. The cellular associations found in these stages and characteristics of the cells used to identify the stages have been well desc ...

The sex of mammalian offspring can be predetermined by flow sorting relatively pure living populations of X- and Y-chromosome-bearing sperm. This method is based on precise staining of the DNA of sperm with the nucleic acid-specific fluorophore, Hoechst 33342, to differentiate between t ...

Charge is a fundamental property of all forms of matter that exhibit attraction or repulsion in the presence of another charged particle. This electrokinetic property occurs when the particles exhibiting a net negative or positive charge are subjected to an external electric field that e ...

Sperm selection is an important part of the ICSI procedure. Routinely, sperm selection for ICSI is based solely on sperm morphology and motility. These latter parameters may not be sufficient to select sperm with intact chromatin. Therefore, sperm selected based on sperm functional chara ...

Annexin-V magnetic-activated cell sorting (MACS) is a new tool to optimize sperm selection in assisted reproduction. The technique is based on the binding of superparamagnetic Annexin-microbeads to externalized phosphatidylserine at the outer leaflet of the plasma membrane of s ...

Approximately 40% of sterility in couples can be attributed to male subfertility and intracytoplasmic sperm injection (ICSI) has become a powerful tool in assisted reproduction to overcome male infertility. Intracytoplasmic morphologically selected sperm injection (IMSI) ...

Enhancement of sperm motility can effectively improve assisted reproductive technique outcomes. Here we describe two (pentoxifylline and platelet-activating factor) popular sperm motility enhancers and their respective methods.