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In Vitro Spermatogenesis Using an Organ Culture Technique

Research on in vitro spermatogenesis has a long history and remained to be an unaccomplished task until very recently. In 2010, we succeeded in producing murine sperm from primitive spermatogonia using an organ culture method. The fertility of the sperm or haploid spermatids was demonstra ...

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Computer-Aided Sperm Analysis (CASA) of Sperm Motility and Hyperactivation

Progressive motility is a vital functional characteristic of ejaculated human spermatozoa that governs their ability to penetrate into, and migrate through, both cervical mucus and the oocyte vestments, and ultimately fertilize the oocyte. A detailed protocol, based on the most com ...

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Manual Methods for Sperm Motility Assessment

Progressive motility is a vital functional characteristic of ejaculated human spermatozoa that governs their ability to penetrate into, and migrate through, both cervical mucus and the oocyte vestments, and ultimately fertilize the oocyte. A detailed protocol, based on traditio ...

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Methods for Direct and Indirect Antisperm Antibody Testing

Antisperm antibodies (ASA) are one well-known cause of refractory infertility in both males and females. In females, a sperm immobilization test, which detects sperm-immobilizing antibodies indirectly in the patient’s serum, requires complement for the reaction and thus seems to be a m ...

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Sperm Morphology Assessment Using Strict (Tygerberg) Criteria

Although sperm morphology evaluation is one of the most important aspects of the semen analysis if done correctly and accurately, a trend is developing in which many laboratories or clinicians no longer regard sperm morphology as relevant due to the very low normal reference value of only 4% morp ...

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Sperm Morphology Classification: A Rational Method for Schemes Adopted by the World Health Organization

Sperm morphology is an important measure of testicular health, spermiation, and fertility potential. The World Health Organization (WHO) Semen Manuals advocate different sperm morphology schemes, but, like the schemes themselves, do not describe classification sequence or ru ...

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The Hypo-osmotic Swelling Test for Evaluation of Sperm Membrane Integrity

A functional membrane is requisite for the fertilizing ability of spermatozoa, as it plays an integral role in sperm capacitation, acrosome reaction, and binding of the spermatozoon to the egg surface. The hypo-osmotic swelling (HOS) test evaluates the functional integrity of the sperm’s ...

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Methods of Sperm Vitality Assessment

Sperm vitality is a reflection of the proportion of live, membrane-intact spermatozoa determined by either dye exclusion or osmoregulatory capacity under hypo-osmotic conditions. In this chapter we address the two most common methods of sperm vitality assessment: eosin–nigrosin ...

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Methods for Sperm Concentration Determination

Proper assessment of the number of spermatozoa is essential not only as an initial step in every clinical infertility investigation but also when attempting to establish the total sperm production in the testis . Reliable methods combined with an understanding of the specific physiology ...

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Evaluation of Skull and Tooth Morphology and Mineralization Using High-Resolution X-Ray Tomography

High-resolution X-ray tomography (microCT) is increasingly available in research settings, and is a valuable tool in the study of mineralized tissue development. At resolutions of 2–20 μm, achievable for typical murine scale samples, it provides nondestructive visualization of th ...

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Organ Cultures and Kidney-Capsule Grafting of Tooth Germs

The study of organogenesis allows investigation of a variety of basic biological processes in the context of the intact organ. The ability to analyze teeth ex vivo during development has emerged as a powerful tool to understand how teeth are constructed and the signaling pathways that regula ...

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Use of siRNA in Dental Tissue-Derived Cell Cultures: Integrin Knockdown in Fibroblasts

Short (or small) interfering RNAs (siRNAs) are double-stranded RNA molecules about 21–25 nucleotides long that have the capacity to disrupt the activity of genes on a posttranscriptional level. This sequence homology-driven gene silencing capacity has been utilized by researchers ...

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Detection of Apoptosis by TUNEL Assay

Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay has been designed to detect apoptotic cells that undergo extensive DNA degradation during the late stages of apoptosis. The method is based on the ability of TdT to label blunt ends of double-stranded DNA br ...

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Immunohistochemistry and Detection of Proliferating Cells by BrdU

Immunohistochemistry is a classic technique used for the localization of antigenic target molecules in �tissue. The method exploits the principle that the target antigen is recognized by specific antibody and is visualized using different detection systems. The subject of this cha ...

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Determination of Gene Expression Patterns by In Situ Hybridization in Sections

In recent years, in situ RNA hybridization technique has found a widespread application in developmental biology. This method has frequently been used to determine gene expression patterns, which is a first step toward understanding of a gene function. Here, we provide a reliable and sensi ...

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Determination of Gene Expression Patterns by Whole-Mount In Situ Hybridization

Whole-mount in situ hybridization (WISH) is a reliable and specific method to study three-dimensional patterns of gene expression. A labeled nucleic acid probe anneals to a complementary target sequence and is visualized and localized in the embryo. This chapter describes a sensitive m ...

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Histological Analysis of the Embryonic and Adult Tooth

Histology is the study of the microscopic anatomy of tissues by examining a thin slice of the tissue under the microscope. Prior to slicing/sectioning, most tissues require some form of solidifying to allow thin sections to be cut. However, since the tooth is the hardest substance in the vertebrate ...

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Whole Mount Immunohistochemistry and In Situ Hybridization of Larval and Adult Zebrafish Dental Tissues

Tooth development is increasingly being studied in a variety of vertebrate model organisms, each contributing its own perspective to our understanding of dental diversity. In situ hybridization and immunohistochemistry are well-established and frequently used techniques ...

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In Vitro Studies on Odontogenic Tumors

Ameloblastomas are uncommon benign neoplasms of the jaws. They originate from dental epithelial cells, but they are not capable of mineralizing or forming enamel. The study of these tumors is limited to live tissue collected from patients during scheduled surgery. Ameloblastomas grow s ...

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Generation of a Bioengineered Tooth by Using a Three-Dimensional Cell Manipulation Method (Organ Germ Method)

The arrangement of cells within a tissue plays an essential role in organogenesis, including tooth development. Organ morphogenesis and physiological functions induced by three-dimensional tissue organization are well known to be regulated by the proper spatiotemporal orga ...

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