Generation of a dual-color reporter mouse line to monitor spermatogenesis in vivo

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.

Paternal H3K4 methylation is required for minor zygotic gene activation and early mouse embryonic development

“…For this particular experiment, we utilized a transgenic (Tg) mouse line which carries a histone H4-Venus fusion transgene under the regulation of an endogenous histone H4 promoter (Fig 3C) [19]. We found weak but substantial expression of H4-Venus in PN4-5 zygotes obtained from mating pairs of H4-Venus homozygous Tg sperm × WT oocytes (Fig 3D).…”

“…To further confirm the inhibitory effect of the H3.3 K4M mutant on paternal transcription during minor ZGA, the mRNA level of a paternally transcribed gene was compared between H3.3 wild-type (WT) versus K4M injection conditions. For this particular experiment, we utilized a transgenic (Tg) mouse line which carries a histone H4-Venus fusion transgene under the regulation of an endogenous histone H4 promoter ( Fig 3C) [19]. We found weak but substantial expression of H4-Venus in PN4-5 zygotes obtained from mating pairs of H4-Venus homozygous Tg sperm × WT oocytes ( Fig 3D).…”

“…Four-to eight-week-old BDF1 mice (C57BL/6 × DBA/2 strain; CLEA Japan, Tokyo, Japan) were used for all experiments. H4-Venus transgenic mice were prepared as previously reported [19]. MII oocytes collected from superovulated female mice were cultured in HTF medium with 3 mg/ml bovine serum albumin.…”

“…H4-Venus transgenic mice were prepared as previously reported [19]. MII oocytes collected from superovulated female mice were cultured in HTF medium with 3 mg/ml bovine serum albumin.…”

Neonatal testis growth recreated in vitro by two‐dimensional organ spreading

“…Histone H4-venus (H4V) transgenic mice (Makino et al, 2013; genetic background: C57BL/6 J-ICR mixed) and Sox9-EGFP knock-in mice (Nakamura et al, 2011;Nel-Themaat et al, 2009;genetic background: C57Bl/6J-129/SvEv-Swiss-ICR mixed) were used. The H4V signal is localized in the nucleus of germ cells from spermatogonia to spermatids, appearing as numerous green dots in the seminiferous tubules when observed with a stereomicroscope.…”