Abstract
In vivo fluorescent imaging technique is a strong tool to visualize the various cellular events such as the proliferation, differentiation, migration, and a lineage tracing in living cells requiring no further experimental procedure such as immunostaining. During spermatogenesis, unique and dynamic histone exchanges occur. Since the timing and types of histone exchanges defines the particular stages of spermatogenesis, visualizing certain types of histones in testes is useful not only for researching specific histone dynamics, but also for monitoring the stages of spermatogenesis in vivo. In this study, we report the establishment of two transgenic (Tg) mouse lines expressing histone H4-Venus (H4V) and histone H3.3-mCherry (H33C) fusion proteins in testicular germ cells, and demonstrated their utility for monitoring germ cell development in vivo. Because of the choice of promoter as well as the nature of these histones, H4V and H33C were exclusively expressed in the germ cells of the distinct stages, which allowed the determination of spermatogenic stages in real time. In addition, disappearance of H4V and H33C at particular stages of differentiation/fertilization also represented dynamic histone removal. Collectively, these Tg mice are a valuable resource not only for monitoring differentiation stages, but also for studying the chromatin dynamics of post-natal testicular germ cell development in vivo.
Highlights
Spermatogenesis is the process of differentiation from spermatogonia to spermatozoa
GENERATION OF TRANSGENIC (Tg) MOUSE LINES To generate H4V transgenic mice, we utilized a Bacterial artificial chromosome (BAC) system to maintain the endogenous expression pattern and level of the H4 histone, as ectopic and excess expression of transgene might be toxic to cells
The DNA fragment encoding a Prm-H3.3-mCherry sequence was subcloned into the Piggybac system vector pBbsr2 (Figure 1B), which enabled us to generate a transgenic line in a transposasedependent manner (Sumiyama et al, 2010; Yusa et al, 2011)
Summary
Postnatal spermatogenesis originates from spermatogonial/spermatogenic stem cells (SSCs), which continuously undergo self-renewal as well as asymmetrical cell division to produce daughter cells, called spermatogonia. These cells proliferate by mitosis, and become spermatocytes, which enter meiotic stages. Most of the histones are gradually eliminated from chromatin, and replaced first by transition proteins (TNPs) and by protamines, both of which are highly basic male germ cell-specific proteins. These steps make the spermatozoal chromain highly condensed, and are necessary for the acquisition of fertility. A recent study suggests transgenerational effects are caused by the retained histones as well as their modifications (Hammoud et al, 2009)
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