RESUMEN
We previously reported the successful induction and completion of mouse spermatogenesis by culturing neonatal testis tissues. The culture medium consisted of α-minimum essential medium (α-MEM), supplemented with Knockout serum replacement (KSR) or AlbuMAX, neither of which were defined chemically. In this study, we formulated a chemically defined medium (CDM) that can induce mouse spermatogenesis under organ culture conditions. It was found that bovine serum albumin (BSA) purified through three different procedures had different effects on spermatogenesis. We also confirmed that retinoic acid (RA) played crucial roles in the onset of spermatogonial differentiation and meiotic initiation. The added lipids exhibited weak promoting effects on spermatogenesis. Lastly, luteinizing hormone (LH), follicle stimulating hormone (FSH), triiodothyronine (T3), and testosterone (T) combined together promoted spermatogenesis until round spermatid production. The CDM, however, was not able to produce elongated spermatids. It was also unable to induce spermatogenesis from the very early neonatal period, before 2 days postpartum, leaving certain factors necessary for spermatogenic induction in mice unidentified. Nonetheless, the present study provided important basic information on testis organ culture and spermatogenesis in vitro.
Asunto(s)
Técnicas de Cultivo de Órganos/métodos , Espermatogénesis , Factores de Edad , Animales , Bovinos , Medios de Cultivo/química , Hormona Folículo Estimulante/farmacología , Técnicas In Vitro , Lípidos/farmacología , Hormona Luteinizante/farmacología , Masculino , Meiosis/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Transgénicos , Albúmina Sérica Bovina/aislamiento & purificación , Albúmina Sérica Bovina/farmacología , Transducción de Señal , Espermatogénesis/efectos de los fármacos , Espermatogénesis/fisiología , Espermatogonias/efectos de los fármacos , Testículo/citología , Testículo/efectos de los fármacos , Testículo/fisiología , Testosterona/farmacología , Tretinoina/farmacología , Triyodotironina/farmacologíaRESUMEN
Although recent findings suggest that the F-box genes SFB/SLF control pollen-part S specificity in the S-RNase-based gametophytic self-incompatibility (GSI) system, how these genes operate in the system is unknown, and functional variation of pollen S genes in different species has been reported. Here, we analyzed the S locus of two species of Maloideae: apple (Malus domestica) and Japanese pear (Pyrus pyrifolia). The sequencing of a 317-kb region of the apple S9 haplotype revealed two similar F-box genes. Homologous sequences were isolated from different haplotypes of apple and Japanese pear, and they were found to be polymorphic genes derived from the S locus. Since each S haplotype contains two or three related genes, the genes were named SFBB for S locus F-box brothers. The SFBB genes are specifically expressed in pollen, and variable regions of the SFBB genes are under positive selection. In a style-specific mutant S haplotype of Japanese pear, the SFBB genes are retained. Apart from their multiplicity, SFBB genes meet the expected characteristics of pollen S. The unique multiplicity of SFBB genes as the pollen S candidate is discussed in the context of mechanistic variation in the S-RNase-based GSI system.
Asunto(s)
Genes de Plantas , Malus/genética , Pyrus/genética , Secuencia de Aminoácidos , Secuencia de Bases , Cromosomas Artificiales Bacterianos/genética , ADN de Plantas/genética , Proteínas F-Box/genética , Haplotipos , Japón , Datos de Secuencia Molecular , Mutación , Filogenia , Proteínas de Plantas/genética , Polen/genética , Polimorfismo Genético , Ribonucleasas/genética , Homología de Secuencia de AminoácidoRESUMEN
Gametophytic self-incompatibility in Rosaceae, Solanaceae, and Scrophulariaceae is controlled by the S locus, which consists of an S-RNase gene and an unidentified "pollen S" gene. An approximately 70-kb segment of the S locus of the rosaceous species almond, the S haplotype-specific region containing the S-RNase gene, was sequenced completely. This region was found to contain two pollen-expressed F-box genes that are likely candidates for pollen S genes. One of them, named SFB (S haplotype-specific F-box protein), was expressed specifically in pollen and showed a high level of S haplotype-specific sequence polymorphism, comparable to that of the S-RNases. The other is unlikely to determine the S specificity of pollen because it showed little allelic sequence polymorphism and was expressed also in pistil. Three other S haplotypes were cloned, and the pollen-expressed genes were physically mapped. In all four cases, SFBs were linked physically to the S-RNase genes and were located at the S haplotype-specific region, where recombination is believed to be suppressed, suggesting that the two genes are inherited as a unit. These features are consistent with the hypothesis that SFB is the pollen S gene. This hypothesis predicts the involvement of the ubiquitin/26S proteasome proteolytic pathway in the RNase-based gametophytic self-incompatibility system.