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The clinical application of human adipose-derived mesenchymal stem cells (MSCs) as treatment for intractable diseases or traumatic tissue damage has attracted attention. To address the ability of reactivating injured ovaries, we prepared a rat model with damaged ovaries by using an anticancer agent, cyclophosphamide (CTX). We then investigated the restorative effects on ovarian function and the safety of adipose-derived MSCs (A-MSCs). MSCs were shown to be capable of inducing angiogenesis and restoring the number of ovarian follicles and corpus lutea in ovaries. No deformities, tumor formation or deaths were observed in F1 and F2 rats, indicating that the local injection of MSCs into the ovary did not have any obvious side effects. In addition, the localization of the Y chromosome was investigated using the fluorescent in situ hybridization method by injecting male A-MSCs into the ovaries; as a result, the Y chromosomes were localized not in the follicles, but in the thecal layers. ELISA revealed that A-MSCs secreted higher levels of vascular endothelial cell growth factor (VEGF), insulin-like growth factor-1 (IGF-1) and hepatocyte growth factor (HGF) than tail fibroblast cells. Quantitative real-time PCR and immunohistochemistry showed that higher expression levels of VEGF, IGF-1 and HGF were observed in CTX-treated ovaries after A-MSC transplantation. These findings suggest that MSCs may have a role in restoring damaged ovarian function and could be useful for regenerative medicine.

Aim: It is difficult to induce ovulation in patients with polycystic ovary syndrome (PCOS) resistant to clomiphene citrate (CC) because of the narrow safety margin of human menopausal gonadotropin preparations. In the present study, patients diagnosed as having PCOS according to the Japanese diagnostic criteria were divided into two groups (Groups H and N). Testosterone level was used as a cut-off value between Groups H and N to examine the usefulness of laparoscopic ovarian drilling (LOD) to induce ovulation. Methods: Thirty-two women with PCOS resistant to CC who underwent LOD were evaluated. The patients were divided into a high and a normal androgen group (Groups H and N with 19 and 13 patients, respectively) based on the level of testosterone measured 1 week before surgery using a cut-off value of 50 ng/dL. LOD was performed with microlaparoscopy under intravenous anesthesia. Results: After LOD, spontaneous ovulation occurred in 16 (84.2%) and nine (69.2%) patients in Groups H and N, respectively. Additional treatment with CC was given to 12 (63.2%) and 11 (84.6%) patients, respectively. No significant difference was noted with regard to the spontaneous ovulation rate after LOD. Eighteen pregnancies were established in 17 patients (53.1%). The pregnancy rate after LOD was not significantly different between Groups H and N: 42.1% (8 patients) and 76.9% (10 patients), respectively. Conclusion: Patients with PCOS as diagnosed based on the Japanese diagnostic criteria include those with increased and normal androgen levels. LOD is considered an effective ovulation induction method for both types of patients with PCOS.

In oocytes, cytoplasmic 3' polyadenylation regulates translational activation of dormant mRNA during meiotic maturation. Thus exogenous proteins are hardly expressed after injection of conventional RNA. To circumvent this, we synthesized a long polyadenylated (approximately 250 A) tail to encode RNA with an enhanced yellow fluorescent protein targeted to mitochondria (EYFP-mito), and injected it into mouse oocytes at the germinal vesicle (GV) stage. From this transcript, EYFP-mito was clearly expressed in approximately 80% of oocytes, while scarce expression from a transcript with only 30 A was observed. In strongly expressing oocytes, fluorescence was detected within 1-3 h after RNA injection, increased linearly up to 12 h, and reached a maximum at 12-15 h. The distribution of EYFP-mito matched the staining of mitochondria in these oocytes. About 80% of these oocytes underwent GV breakdown and 60% matured in vitro, comparable to non-expressing or non-RNA-injected oocytes. Some of the oocytes which strongly expressed EYFP-mito remained at the GV stage. Thus, the expression was not always accompanied by meiotic maturation, nor did it suppress the maturation process. Mature oocytes expressing EYFP-mito possessed normal fertilizability associated with intracellular Ca(2+) oscillations, and developed into 2-cell embryos. Thus, polyadenylated RNA is a useful tool applicable to the expression of EYFP-fused functional proteins or of indicator protein probes for studies of mammalian fertilization.