Hirotaka Matsumi

Stem cell factor (SCF) concentrations in peritoneal fluid of women with or without endometriosis.

PROBLEM: In the quest for possible involvement of stem cell factor (SCF), a cytokine known to have multiple effects, in the pathogenesis of endometriosis, we evaluated concentrations of SCF in peritoneal fluid (PF) of women with or without endometriosis.
 METHOD OF STUDY: SCF concentrations in PF collected from women undergoing laparoscopy were measured, using a specific enzyme‐linked immunosorbent assay (ELISA). Reverse transcription‐polymerase chain reaction (RT‐PCR) analysis to detect gene expression of c‐kit, the receptor for SCF, was performed using the endometriotic tissue and the eutopic endometrium collected during the operation.
 RESULTS: SCF concentrations in PF of women with endometriosis were significantly higher compared to women without endometriosis. Looking at SCF concentrations in PF of women with endometriosis stratified by disease stage, women with stage I and II exhibited relatively higher SCF levels in PF, whereas SCF levels in PF with stage III and IV were comparable with those without endometriosis. The expression of mRNA for c‐kit was detected in both the endometriotic tissue and the eutopic endometrium.
 CONCLUSION: We demonstrated an elevation in SCF levels in PF associated with endometriosis and the presence of its receptor in endometriotic tissues. Given the known pleiotropic properties of SCF, the present results suggest that SCF might play a role in the pathogenesis of endometriosis.

Effect of Luteinizing Hormone-Releasing Hormone Analogs on the Rat Ovarian Follicle Development

Our objective was to study the direct action of luteinizing hormone-releasing hormone (LHRH) agonist buserelin and LHRH antagonist Cetrorelix (SB-75) on cell proliferation and differentiation in the rat ovarian follicle. Preovulatory follicles were isolated from PMSG-primed immature rats and incubated in the presence or absence of hCG (10 IU/ml), buserelin (10(-9)-10(-6) M) or Cetrorelix (10(-9)-10(-6) M) for 12 h in vitro. Buserelin induced meiotic maturation of the follicle-enclosed oocytes dose-dependently. The percentage of oocytes with germinal vesicle breakdown at 10(-6) M buserelin (73.3%) did not differ from that of hCG-treated control (73.3%). Buserelin also significantly stimulated prostaglandin E2 and progesterone production by follicles, but not estradiol production. Granulosa cells were obtained from the preovulatory follicles and cultured for 5 days. Epidermal growth factor (EGF) stimulated granulosa cell growth at concentrations of 1, 10 and 100 ng/ml. In contrast, both buserelin and Cetrorelix inhibited granulosa cell growth dose-dependently in the range of 10(-10)-10(-5) M, with Cetrorelix inducing a greater growth inhibition than buserelin. Electrophoretic analysis of genomic DNA extracted from granulosa cells treated with 10(-6) M concentration of either LHRH analog revealed a definitive ladder pattern of oligonucleosomal length DNA fragments characteristic of apoptosis. Western blotting detected that EGF-induced tyrosine phosphorylation was not affected by either analog. These results demonstrate that LHRH agonist and antagonist inhibit directly proliferation of granulosa cells through apoptosis, without interference with EGF receptor phosphorylation, whereas LHRH agonist stimulates cell differentiation in the preovulatory follicle.

Regulation of Nitric Oxide Synthase to Promote Cytostasis in Ovarian Follicular Development

Abstract Our own recent studies have demonstrated that inducible nitric oxide synthase (iNOS) is predominantly localized in granulosa cells of healthy immature follicles in the rat ovary, whereas granulosa cells of either healthy mature follicles or follicles destined to be atretic are devoid of iNOS. These findings suggest that iNOS is pivotal for immature follicles to remain dormant. To test this hypothesis, we examined the effects of a GnRH agonist (buserelin), a proapoptotic substance, and epidermal growth factor (EGF), a mitogenic and, consequently, antiapoptotic factor, on the amount of iNOS mRNA in rat granulosa cells. Administration of buserelin in immature female rats transiently diminished iNOS mRNA levels in the ovaries as determined by Northern blot analysis. In cultured rat granulosa cells, buserelin and EGF increased the incidence of apoptosis and DNA synthesis, respectively, whereas both reduced iNOS mRNA levels as determined by reverse transcription-coupled polymerase chain reaction. The concomitant addition of S-nitroso-N-acetyl-dl-penicillamine, an NO donor, together with buserelin or EGF eliminated the observed effects of these substances (i.e., induction of apoptosis and stimulation of DNA synthesis, respectively). These results suggest that the changes in developmental status of immature follicles either into development or atresia are associated with reduced iNOS levels in granulosa cells, thus reinforcing the notion of NO as a cytostatic factor in ovarian follicles.

Estriol add-back therapy in the long-acting gonadotropin-releasing hormone agonist treatment of uterine leiomyomata

The hypoestrogenic state induced by gonadotropin-releasing hormone agonists (GnRHa) has been shown to be effective in the treatment of uterine leiomyomas but to induce bone loss. Estriol has been described to be a weak and short-acting estrogen without an increased risk of endometrial proliferation and hyperplasia. The purpose of this study was to evaluate whether treatment of uterine leiomyomata with GnRHa plus oral estriol add-back therapy could prevent bone loss, without deteriorating the therapeutic effect of GnRHa. Twelve premenopausal women with symptomatic uterine leiomyomas were randomized to receive either leuprolide acetate depot alone at a dose of 3.75 mg s.c. every month for 6 months (non add-back group; n = 6), or GnRHa for 6 months plus oral estriol 4 mg/day for 4 months commencing with the third GnRHa injection (add-back group; n = 6). In the add-back group, leiomyoma volume, as measured by transvaginal ultrasound, decreased to 59.1% of baseline at 2 months of GnRHa therapy with no significant change in size during the remaining treatment period. In contrast, it decreased to 31.3% of pretreatment size at the end of treatment in the non add-back group. The levels of bone metabolic markers such as CrossLaps, deoxypyridinoline, osteocalcin and bone-specific alkaline phosphatase, increased significantly throughout the treatment in the non add-back group, whereas they were suppressed by the add-back therapy. The bone mineral density of lumbar spine (L2-L4) as measured by dual-energy X-ray absorptiometry decreased significantly by 7.5% at the end of treatment in the non add-back group, but did not change significantly in the add-back group. In conclusion, GnRHa plus estriol add-back therapy might be considered for long-term treatment of uterine leiomyomata.

Effects of gonadotropin-releasing hormone analog treatment on skin condition.

The skin is a target organ of estrogens. Thus, theoretically, a hypoestrogenic state induced by gonadotropin-releasing hormone analog (GnRHa) treatment may have effects on skin condition. The aim of this study was to evaluate skin condition during GnRHa treatment. Sixteen premenopausal women undergoing GnRHa treatment for 16 weeks, as a presurgical treatment for uterine leiomyomas, were studied. Measurement of serum estradiol levels and epidermal hydration, and evaluation of subjective findings on skin condition using a questionnaire, were performed every 4 weeks during the treatment period. Serum estradiol levels were significantly suppressed at 4 weeks of treatment, and remained low afterwards. Epidermal hydration measured by corneometer did not show any significant difference at any time point examined, compared with that before treatment. No particular subjective findings relating to the skin (dryness, wrinkling, roughness, pigmentation, itching, formication, reaction to cosmetics) were reported during treatment, whereas complaints about hot flushes and sweating were notable. The results of this preliminary study support the notion that GnRHa treatment for 16 weeks is unassociated with apparent changes in skin condition.

In Situ Hybridization for RNA: Nonradioactive Probe: ds cDNA Probe

As has been shown previously (Koji et al. 1988; 1990; 1996), nonradioactively labeled probes are widely used for in situ hybridization because they have several advantages compared to radioactive ones.