Isolation of Viable Male and Female Gametes in Cunninghamia lanceolata

Viable male and female gametes were isolated from pollinated ovules of Cunninghamia lanceolata (Lamb.) Hook. Prior to their penetration into the female gametophyte, the pollen tubes were drawn out from the nucetlus. The isolated pollen tubes were branched and one of them became swollen. An enlarged spermatogenous cell and subsquently a pair of sperm cells were formed as the pollen tube reached the regions over and against the archegonia. The sperm cells were released from the pollen tubes manually with the use of a stereomicroscope. The positive FDA reaction gave evidence of the sperm cells viability, and the Fluorescent Brightener 28 positive demonstrated the presence of cell wall. The egg cells were enzymatically isolated from the female gametophytes. The isolated egg cells were spherical, contained 1 to 2 large and many small vacuoles. FDA test showed the egg cells were viable, and the viability sustained for 8 days in 2 to 4 ℃ without any protectants. Fusion between single pair of male and female gametic protoplasts was attempted with PEG method, but only adhesion of the two was obtained.     

SIRT1在雌性配子发生中的作用及相关疾病的研究进展

沉默信息调节因子1[Sirtuin (silent mating type information regulation 2 homolog) 1,Sirt1]是依赖于烟酰胺腺嘌呤二核苷酸的组蛋白去乙酰化酶,可与FOX转录因子家族中的O亚家族(FOXOs)、P53等多种转录因子结合。Sirt1通过调控卵巢储备、卵泡形成发育、卵母细胞分化成熟、颗粒细胞功能等生理过程影响雌性生殖系统的生命活动。作者综述了Sirt1对雌性生殖细胞发育及成熟的影响,并总结了Sirt1在多种雌性生殖系统疾病中的研究进展,以期利用其作用机制为提高雌性配子品质和改善雌性生殖机能的相关研究提供理论依据。     

Defining oligosaccharide specificity for initial sperm-zona pellucida adhesion in the mouse

The identity of the sperm surface protein(s) responsible for sperm-zona pellucida binding in the mouse, as well as the characteristics of the oligosaccharide groups on zona pellucida glycoprotein 3 (ZP3) having ligand activity toward this receptor, remain controversial. Conflicting results from several groups have made interpretation of the current data difficult. By developing a quantitative binding assay to evaluate the molecular interactions between mammalian sperm and the zona pellucida during initial gamete interactions, we directly quantified sperm-ZP binding interactions at the molecular level for the first time. The ZP binding assay demonstrated that live, capacitated mouse sperm bind solubilized ¹²⁵I-labeled ZP glycoproteins in a concentration-dependent manner characterized by a rapid forward rate constant of 3.0 × 10⁷ M⁻¹ min⁻¹. Following the initial characterization, the binding assay was used to examine the roles of the sperm surface enzymes galactosyltransferase (GalTase) and fucosyltransferase (FucTase) in sperm-zone pellucida binding in the mouse. These data indicate that substrates for FucTase, but not for GalTase, inhibit sperm-ZP binding, in contrast to earlier reports in which GalTase substrates significantly inhibited sperm binding to intact ZPs. A model is presented which resolves conflicting results between assays using intact ZPs and the results obtained here using soluble ¹²⁵I-ZPs. Assuming a complex binding/recognition site, monosaccharides that could occupy part of the binding site would have a dramatic effect on sperm-ZP binding to the intact ZP, since they need only occupy the binding sites for a short time (∼ 100 msec) to disrupt binding. The current results suggest that the sperm ZP3 receptor binding site minimally recognizes the galβ1,3GlcNAc moiety also recognized by FucTases. The current data do not exclude the possibility that additional sugar residues form part of the ligand oligosaccharide group and are recognized by a yet-to-be-identified sperm surface protein which serves as the ZP3 receptor. © 1996 Wiley-Liss, Inc.     

The reproductive biology of Polytrichum formosum: clonal structure and paternity revealed by microsatellites

Using highly polymorphic microsatellite markers, we assessed clonal structure and paternity in a population of the bryophyte species Polytrichum formosum. Identical multilocus genotypes of individual shoots were almost never observed in spatially separated cushions, but were found to be highly clustered within moss cushions. Therefore, asexual reproduction through dispersal of gametophyte fragments is not very important in P. formosum. However, asexual reproduction on a very localized scale through vegetative growth of genets (branching of gametophytes via clonal growth of rhizomes) is very extensive. The patchy spatial distribution of genets and the absence of intermingling among genets suggest that this species follows a 'phalanx' clonal growth strategy. Vegetative proliferation of genets will increase their size, and, consequently, will have considerable fitness consequences for individuals in terms of increased genet longevity and reproductive output. Although paternity analysis of sporophytes confirmed male genet size, i.e. gamete production, to be an important determinant of male reproductive fitness, it also showed that the spatial distance to female genets is the predominant factor that governs male reproductive success. Moreover, we showed that male gamete dispersal distances in P. formosum are much further than generally assumed, and are in the order of metres rather than centimetres. Combining the findings, we conclude that the high genotypic diversity observed for this facultatively clonal species is most likely explained by a preponderance of sexual reproduction over clonal reproduction.     

High gene flow levels lead to gamete wastage in a desert spider system

Field censuses, breeding experiments, and a quantitative model are used to obtain insight into the extent and consequences of genetic mixing between locally adapted populations of a desert spider. Typically, 9% of the matings of desert riparian spiders (non-aggressive phenotype) in native habitat involve an arid-land partner (aggressive phenotype). Mating was found to be random with respect to behavioral phenotype, but linearly related to both the rate of immigration and survival of immigrants from surrounding arid habitats in the riparian area. Genetic mixing between riparian and arid-land spiders produces offspring that exhibit lower rates of survival in riparian habitat. Two extreme behavioral phenotypes were also observed in the field studies: approximately 5% of the female spiders attacked all males they encountered while another 22% ran from all potential mates. Punnett square analyses of the potential genotypes produced by introgression between arid- and riparian-adapted spiders indicate that these extreme phenotypes appear in F2 generation hybrids and backcrosses. Because there is a costly wastage of gametes in the case of mixed phenotype mating, model results indicate that within three generations of the cessation of gene flow, the riparian population would be free of mixed genotypes and moving towards genetic differentiation.     

Trace elements in the muscle,ova and seminal fluid of key clupeid representatives from the Gdansk Bay (South Baltic Sea) and Iberian Peninsula (North-East Atlantic)

BackgroundBaltic herring and European sardine are pelagic, fish of particular ecological importance, on the one hand control numbers of planktonic organisms, and on the other hand exist as food for predators on higher trophic levels. Moreover, these fish are among the main species caught for human consumption. Rare earth elements (REEs) come mainly from geogenic sources but, due to their use in technology, agriculture and medicine, the importance of anthropogenic sources is growing steadily.MethodsSamples used for the study were available on the market. Fresh materials of fish muscle, ova and seminal fluid were mineralized and elements were determined by means of inductively coupled plasma – mass spectrometry (ICP-MS).ResultsThe conducted research indicated the presence of REEs in the muscles of the Baltic herring (∑REE = 0.076 ± 0.047 mg/kg) and European sardine (∑REE = 0.191 ± 0.163 mg/kg), with a clear dominance of heavy REEs in both fish species. Trace elements (TE) in the muscles of the tested fish demonstrated a similar system of concentration (Baltic herring: Zn > As > Se > Cu > Cr > Ni > Pb > Cd; European sardine: Zn > As > Se > Cu > Ni > Cr > Pb > Cd). REEs and TEs in these fish were presence in ova and seminal fluid indicates intergenerational transfer.ConclusionChanges in the concentrations of some trace elements (As, Cu, Cd) in the muscles of herring indicate increases compared to the historical data. The availability of metals in the aquatic environment may be determined by ongoing climate changes, effected water salinity and warming increased availability of labile forms of trace metals. Decline trends in the condition of pelagic fish need to extend the research in the context of contemporary environmental threats.     

Signal transduction during fertilization in the unicellular green alga, Chlamydomonas

Sexual reproduction in the green alga, Chlamydomonas, is regulated by environmental conditions and by cell–cell interactions. After gametogenesis, flagellar adhesion between gametes triggers gamete activation, leading to cell fusion and zygote formation. Recent studies have identified new molecular events that underlie signal transduction during Chlamydomonas fertilization, including expression of a sex-determining protein, phosphorylation of a homeodomain protein, activity of a kinesin II and regulated translocation of an aurora/Ip11-like protein kinase from the cell body to the flagella.