Heat stress inhibits ovarian follicular development in mammalian species. We hypothesized that heat stress inhibits the function of follicular granulosa cells and suppresses follicular development. To test this, immature female rats were injected with pregnant mare serum gonadotropin (PMSG) at 48 h after the start of temperature treatment (control: 25 8C, 50% RH; heat stress: 35 8C, 70% Relative Humidity). The ovaries and granulosa cells of follicles at different developmental stages were analyzed for gonadotropin receptor levels and aromatase activity; estradiol levels were measured in follicular fluid. Before injection, heat stress diminished only the amount of FSH receptor on granulosa cells of antral follicles. During PMSG-stimulated follicular development, heat stress strongly inhibited gonadotropin receptor levels and aromatase activity in granulosa cells, and estradiol levels in the follicular fluid of early antral, antral and preovulatory follicles. To examine apoptosis and mRNA levels of bcl-2 and bax in granulosa cells, follicles harvested 48 h after PMSG injection were cultured in serum-free conditions. Heat-stressed granulosa cells showed a time-dependent increase in apoptosis. The bcl-2 mRNA levels were similar in control and heat-stressed granulosa cells; bax mRNA levels were increased in heat-stressed granulosa cells. According to these results, heat stress inhibits expression of gonadotropin receptors in granulosa cells and attenuates estrogenic activity of growing follicles, granulosa cells of heat-stressed follicles are susceptible to apoptosis, and the bcl2/bax system is not associated with heat-stress-induced apoptosis of granulosa cells. Our study suggests that decreased numbers and function of granulosa cells may cause ovarian dysfunction in domestic animals in summer.

γ-aminobutyric acid (GABA) is a four-carbon non-protein amino acid that is present in a wide variety of prokaryotic and eukaryotic organisms. Because of its antihypertensive effect on the human body, the demand for naturally occurring GABA has increased recently in the Japanese food industry. In this study, we evaluated the GABA content of tomato fruits of 61 commercial cultivars, wild species, and wild derivatives in 2005 and 2006 to screen for resources to breed a GABA-rich cultivar. GABA contents in tomato fruits greatly varied among the tested varieties and were poorly reproducible between the tested years. GABA-rich candidates selected from the screening were then subjected to salinity-stressed cultivation using the NFT system to assess their suitability for cultivation to produce GABA-rich fruit. Based on the results of two screenings and the salinity-stress cultivation test, 'DG03-9' was selected as a GABA-rich cultivar. The accumulation profiles for GABA, glutamine, and glutamic and aspartic acid during fruit development were also investigated in 'DG03-9' and 'House Momotaro' under salinity stress. The GABA content peaked at 24 days after flowering (DAF) in 'DG03-9' and 36 DAF in 'House Momotaro', and then declined during ripening. Salinity stress apparently promoted GABA accumulation during the early developing stages, but its effect on GABA decrease was different between the varieties. Although the GABA content in red mature fruits of 'DG03-9' was higher than that in fruits of 'House Momotaro' under normal and saline conditions, the maximum contents were almost the same in both cultivars. These results suggest that the lower reduction rate of GABA during the ripening stage causes high GABA accumulation in 'DG03-9' fruit. This variety will be a useful resource in the breeding of new GABA-rich cultivars. Additionally, we utilized an enzymatic assay with GABase to quantify GABA content in tomato fruit. This method will be a powerful screening tool for breeding.Key Words: γ-aminobutyric acid (GABA), salinity stress, Solanum lycopersicum, tomato.Introduction γ-aminobutyric acid (GABA) is a ubiquitous fourcarbon non-protein amino acid that is present in a wide range of organisms, including bacteria, vertebrates, and plants. In animals and plants, GABA is mainly metabolized via a short three-enzyme pathway termed the "GABA shunt", a bypass of the tricarboxylic acid (TCA) cycle (Bouché and Fromm, 2004;Shelp et al., 1999). In this shunt, GABA is irreversibly synthesized from glutamic acid by glutamic acid decarboxylase (GAD) (Chung et al., 1992;Tuin and Shelp, 1994) and reversibly converted to succinic semialdehyde by GABA transaminase (GABA-T) (Bouché and Fromm, 2004;Shelp et al., 1999). Succinic semialdehyde is then irreversibly reduced by succinate semialdehyde dehydrogenase (SSADH), and the resulting succinate eventually flows back into the TCA cycle.In plants, GABA and the GABA shunt play roles in various physiological processes, including nitrogen storage (Breitkreuz et al., 1999), regulation of cytosol...

Effects of heat stress on follicular development was studied in three-week-old immature female rats kept under 25℃ and 50% relative humidity (RH). In experiment 1, animals were divided