Gene expression profile during ovarian folliculogenesis.

Assisted reproductive technologies have progressed significantly and have provided successful treatment for many infertile couples. However, more advanced technologies are required for severe infertility such as premature ovarian failure and ovarian impairment due to adjuvant therapy for cancer. Ovarian tissue cryopreservation followed by in vitro growth of isolated follicles is a feasible proposition for such patients. Close coordination of communication among follicle cells including oocytes, granulosa and theca cells is required for follicle growth. Crucial factors may regulate the gonadotropin-independent and -dependent follicle growth stages. To facilitate development of a culture system for early growing follicles, DNA microarray analysis of mouse ovaries recovered at 7, 10, 13, 16 and 19 days of age was performed to identify factors required for the growth of early-stage follicles. These studies showed strong intensity of zona pellucida glycoproteins, bone morphogenic protein-15 (BMP-15) and growth differentiation factor (GDF-9) in 7 days old mice, which gradually declined in 19 days old mice. KIT, KIT ligand, anti-müllerian hormone (AMH) and platelet-derived growth factor (PDGF), known as granulosa cell secreted factors, also showed relatively high expression. These studies will facilitate our understanding of the regulatory factors involved in folliculogenesis and thereby enable establishment of in vitro culture system for ovarian follicles.

Limited effect of chromatin remodeling on D(beta)-to-J(beta) recombination in CD4+CD8+ thymocyte: implications for a new aspect in the regulation of TCR beta gene recombination.

We have generated mutant mice in which TCR beta chain enhancer (E(beta)) was replaced with the TCR alpha chain enhancer (E(alpha)). Using this mouse model, we analyzed (i) recombination status of the TCR beta chain genes after functional V(D)J rearrangements occurred in the first allele during double-negative (DN)-to-double-positive (DP) transition and (ii) involvement of E(beta) for the expression of rearranged TCR beta chain genes. Our data show that E(alpha) substituted for E(beta) function to express a similar extent of TCR beta chains exactly at the same time as did E(beta) (CD25+CD44- DN stage), although the proportion of TCR beta+ cells at this stage was low in mutant mice. At the DP stage, germline transcription and histone acetylation of D(beta)-J(beta) loci were detectable at a high degree in both mutant and wild-type mice. However, DP cells in mutant mice retained the germline D(beta)-J(beta) configuration at a higher frequency than that of wild-type mice, whereas both DP cells expressed TCR beta chains to a similar extent. These data suggest that chromatin opening has a limited impact on D(beta)-to-J(beta) recombination at the DP stage and that E(alpha) is functionally equivalent to E(beta) in promoting expression of functionally rearranged TCR beta chain genes through DN-to-DP transition.

Evaluation of recombinant interleukin-2 immunotherapy for human hemangiosarcoma in a SCID mice model (WB-SCID).

We treated the patients with cutaneous hemangiosarcoma with recombinant interleukin-2 (rIL-2) immunotherapy that showed clear therapeutic effects. This immunotherapy is popular for the treatment of hemangiosarcoma in Japan. The purpose of this study is to clarify the clinical effects in an animal experiment. After establishing a SCID mouse model of human hemangiosarcoma WB-SCID, we used this model to investigate anti-tumor effects of rIL-2 and LAK cells. We demonstrated that hemangiosarcoma cells are LAK-sensitive, and LAK cells induced by rIL-2 suppress the growth of hemangiosarcoma. Our results may assure the clinical effects of rIL-2 immunotherapy on hemangiosarcoma.