Impaired active demethylation of the paternal genome in pronuclear-stage rat zygotes produced by in vitro fertilization or intracytoplasmic sperm injection.

This study was designed to investigate the effect of in vitro production systems of rat zygotes such as in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) on demethylation dynamics of the paternal genome. Immunostaining with anti-5-methylcytosine indicated that in vivo derived zygotes harvested at 20 hr post-hCG injection had no active demethylation of paternal genomes as a mean relative methylation (RM) value, total fluorescence in the paternal genome divided by that in maternal one, at 1.17. Leaving zygotes in vivo or in culture for an additional 4 or 8 hr resulted in significant decreases of RM values to 0.14-0.31. Since in vitro-derived zygotes were produced using oocytes harvested at 14 hr post-hCG injection, zygotes at 6 hr after IVF or ICSI were considered developmentally comparable to the in vivo derived zygotes harvested at 20 hr post-hCG injection, with RM values at 1.04 and 0.92, respectively. At 10 hr post-IVF and ICSI, the RM values of the in vitro derived zygotes decreased significantly, but to a lesser extent compared with in vivo derived zygotes, to 0.53 and 0.62, respectively, without further decreases at 14 hr. Treatment of IVF-derived zygotes with 5-aza-2'-deoxycytidine (5-azadC; an inhibitor for methylation) or trichostatin A (TSA; an inhibitor for deacetylation) resulted in the decreased RM values at 14 hr post-IVF. However, developmental potential of the 5-azadC- or TSA-treated IVF zygotes up to the blastocyst stage was not improved. Thus, the demethylation dynamics of the paternal genome in pronuclear-stage rat zygotes was impaired by routine protocols for in vitro embryo production such as IVF and ICSI.

Active demethylation of paternal genome in mammalian zygotes.

Epigenetic reprogramming in early preimplantation embryos, that refers to erasing and remodeling epigenetic marks such as DNA methylation, is essential for differentiation and development. In many species, paternal genome is subjected to genome-wide active demethylation before the DNA replication commences, while maternal genome maintains its methylation status until being demethylated passively during the subsequent cleavage divisions. The purpose of this manuscript was to review the available knowledge about the paternal genome active demethylation process concerning the possible mechanisms, species variation and the factors affecting the active demethylation dynamics such as in vitro protocols for production of pronuclear-stage zygotes. Better understanding the mechanisms by which the epigenetic reprogramming is occurred may contribute to clarify the biological significance of this process.

Availability of subfertile transgenic rats expressing the c-myc gene as recipients for spermatogonial transplantation.

The spermatogonial transplantation system was applied to evaluate stem cell kinetics and niche quality and to produce gene-modified animals using the stem cells after homologous recombination-based selection. This study was designed to determine whether the transplanted spermatogonia were able to proliferate and differentiate in male rats expressing the c-myc transgene under control of the human metallothionein IIA promoter (MT-myc Tg rats). Donor testicular cells were prepared from heterozygous chicken beta actin (CAG)/enhanced green fluorescent protein (EGFP)-transgenic rats (EGFP Tg rats) during the second week after birth and injected into the seminiferous tubules of the MT-myc Tg rats (line-A and -B; both subfertile) or rats pretreated with busulfan to remove endogenous spermatogonia. Three to four months after transplantation, cell colonies with EGFP fluorescence were detected in 36% (4/11), 40% (8/20), and 71% (5/7) of the transplanted testes in line-A MT-myc Tg rats, line-B MT-myc Tg rats, and busulfan-treated rats, respectively. No EGFP-positive colonies were detected when wild-type male rats were used as recipients (0/7; testis-basis). The histopathological and immunofluorescent examination of the serial sections from the transplanted testes showed normal spermatogenesis of the donor spermatogonia, but atrophy of the recipient seminiferous tubules. Microinsemination with round spermatids and mature spermatozoa derived from EGFP-positive testes in line-A rats resulted 26% (10/39 transferred) and 23% (11/48 transferred) full-term offspring, respectively. Thus, the MT-myc Tg male rats were suitable as potent recipients for spermatogonial transplantation without any chemical pretreatment to remove the endogenous spermatogonia.

Optimal timing of MR sialography by use of a simple method of stimulating the salivary gland: a preliminary report.

The present study was performed for determining the optimal timing of MR sialography by use of the Japanese pickled plum (umeboshi) for promoting secretion by the salivary glands. MR sialography was performed in four healthy male volunteers. The four volunteers were examined before and 10 min after stimulation with umeboshi. On the next examination, three volunteers were examined before and after umeboshi stimulation every 1 min up to 5 min to allow assessment of the temporal changes in duct visualization. Dilatation of the salivary gland ducts and improvement of the visualization of the ducts were obtained after stimulation with umeboshi. The difference in the dilatation of the parotid duct was statistically significant. In the temporal study, the salivary gland ducts were shown to be dilated at 2 min after stimulation. As a result, 2 min after stimulation is the optimal timing for MR sialography by use of umeboshi as a stimulator of salivary secretion.

A case of hypereosinophilic syndrome.

A 22-year-old man developed papules on his legs in November of 2001, which then spread to cover almost his entire body. He was treated with a topical steroid and PUVA therapy at another hospital. The symptoms showed no improvement, and, in February of 2002, he came to our hospital. Examination revealed hypereosinophilia, and, because he had symptoms of organ involvement by the heart, lung, and inguinal lymph nodes as well as the skin, we diagnosed him with idiopathic hypereosinophilic syndrome (HES). The patient was treated with oral prednisolone at a dose of 60 mg/day, and his cutaneous lesions and other organ symptoms improved.

Water, salts and skin barrier of normal skin.

BACKGROUND: We recently reported that open application of seawater for 20 min ameliorated experimental irritant contact dermatitis induced by sodium lauryl sulphate (SLS) cumulative irritation. The efficacy was overall contributed by 500 mm of sodium chloride (NaCI) and 10 mm of potassium chloride (KCl), which are consistent with the each concentration in seawater. Although the usefulness of mineral water with 500 mm NaCl and 10 mm KCl to treat atopic dermatitis (AD) or irritated skin was considered, seawater or its components would induce a feel of stinging in patients with AD. Furthermore, 20 min application was thought to be too long to use everyday as a treatment. OBJECTIVE: We report the effects of 3 types of mineral water with NaCl and KCl to check the further efficacy with lesser stinging by 2 min application. RESULTS: A mineral water with 250 mm NaCl and 50 mm KCl was the most effective water to prevent disruption of skin barrier and stratum corneum water content after cumulating irritation by SLS. Moreover, improvement of skin dryness and pruritus were shown 2 weeks after the application of the mineral water to a 6-year-old boy with atopic dermatitis. CONCLUSION: Our results suggested the possible usefulness of the mineral water with 250 mm NaCl and 50 mm KCl as the therapy of atopic dermatitis of other chronic dermatitis. Although the mineral water would not cure those skin diseases, it could be an adjunctive therapy. Further controlled clinical trials with evaluation by TEWL and capacitance are required to declare the efficacy of the mineral water in the treatment of patients with AD or other chronic dermatitis.