Relaxin-like factor (RLF), also known as insulin-like factor 3 (INSL3), is produced by testicular Leydig cells, but its specific receptor LGR8 (leucine-rich repeat family of G-protein-coupled receptor 8) has not been identified in goats. This study aimed to identify complementary DNA (cDNA) sequences of goat LGR8, and characterize the expression of both RLF and LGR8 in goat testes by RT-PCR and immunohistochemistry. Testes were collected from immature (3-month-old) and mature (24-month-old) Saanen goats, and partial cDNA sequences of the goat homologue of human LGR8 were identified. The sequence encoded a reduced peptide sequence of 167 amino acids, which corresponded to transmembrane regions 2 through 5, followed by the beginning of intracellular loop 3 of human LGR8. Expression of both LGR8 and RLF genes was drastically increased in mature testes compared with immature ones. Although RLF protein was restricted to Leydig cells, LGR8 protein was detected in both Leydig cells and seminiferous epithelial cells (possibly germ cells and Sertoli cells). These results reveal a possible existence of the RLF-LGR8 ligand-receptor system within the goat testis, suggesting that RLF may play a role in testicular function through LGR8 on Leydig cells and seminiferous epithelial cells in an autocrine and/or paracrine manner.
Rose is one of the most important floricultural crops in the world and the production of new cultivars in this plant has been a major issue for many years. Modern roses have been mainly developed through spontaneous or artificially induced bud sport1), but the recent progress of plant biotechnology provides the alternative applications such as tissue culture2, 3) and gene manipulation technologies4) for plant breeding. In our laboratory, we have investigated the use of abundant somaclonal variations induced in tissue cultures and attempted to establish an efficient system for the selection of useful genetic variations for improving important traits such as disease resistance in various crop plants5). Also in rose plants, we have elaborated an effective method for tissue culture and clarified the conditions suitable for plant regeneration from the leaf explant-derived calli6). To further develop our culture system, the present work describes an utilization of petal tissues as a culture material for successful callus induction and subsequent plant regeneration in rose plants.Fully opened flowers were harvested from rose plants (Rosa hybrida cv. Carl Red) grown in a greenhouse for 2-3 months and used in the following culture experiment. The receptacles, sepals, and outermost petals were removed from flowers, and the remaining petals were excised and dipped into 70% ethanol for 30 sec and then into a 1 % sodium hypochlorite solution for 90 sec for surfacesterilization. After rinsing with sterilized distilled water, petals were cut into smaller segments (1X1cm) and then placed on a Murashige-Skoog7) (MS) medium supplemented with 3% sucrose and different concentrations of a-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP) and solidified with 0.3% Gelrite (Merk, NJ., USA). The medium was adjusted to pH 5.7 with 1 N NaOH before autoclaving. Culture bottles were tightly sealed with Parafilm and incubated at 26+ 1C in the dark. After 7-10 days of incubation, pale yellow calli were induced from the decolorized edge portions of red-color petal explants of Carl Red in all the combinations of NAA and BAP concentrations ( Fig. 1-A). These calli proliferated slowly, became brownish, and produced adventitious roots (5-10 roots from each petal explant) 14-16 days after incubation ( Fig. 1-B). The root elongation ceased 30-40 days after incubation, and subsequently the bugle-like structures (BLS), as shown in Fig. 1-C, were produced apart from the adventitious roots in the callus tissues approximately 50 days after incubation. The formation of BLS was frequently observed in calli cultured in the presence of 0.25-1.0 ug/ml of NAA and 0.0025-0.015 cg/ml of BAP (Table 1). Especially, the BLS formation was highest in frequency (62% in average of four separate experiments) and in number (15-20 per explant) in combination of 0.75 cg/ml NAA and 0.01, ag/ml BAP. In a previous
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