In vitro analysis of oocyte cumulus complex pickup rate in the hamster Mesocricetus auratus

In mammals, the oocyte and its surrounding cumulus cells constitute an oocyte cumulus complex (OCC). During ovulation, OCCs are extruded into the peritoneal or bursal cavity, depending on the species, and are then rapidly picked up by the fimbria on the outer surface of the oviductal infundibulum and transported to the ampulla, where fertilization occurs. We developed a method to measure OCC pickup rates quantitatively in vitro, and we used this method to evaluate the effects of viscosity and temperature on pickup rates. Hamster infundibula are placed in a holding pipette in a chamber modified to study OCC pickup. Ciliary beat frequencies (CBF) can be measured in the same preparation. Pickup rates vary depending on the pathway on which the OCC travels over the surface of the infundibulum; however, rates for a given pathway are very consistent. The average pickup rate at room temperature calculated from three different pathways/infundibulum was 55.2 ± 10.6 μm/sec. Both rates between infundibula from the same female and rates among infundibula from different females were in most cases similar. Preparations preincubated in vitro for 2.75 hr produced rates similar to nonpreincubated samples, while longer preincubation resulted in decreased rates. Inclusion of Ficoll in culture medium to increase viscosity caused a concentration-dependent decrease in both OCC pickup rate and in CBF. However, a significant decrease in OCC pickup rate was only observed at viscosities higher than those found in bursal fluid. When trials were run at physiological temperature (36.4°C) rather than ambient temperature, rates increased to 136.7 ± 29.9 (SD) μm/sec. Linear regression analysis demonstrated a strong positive correlation (r = 0.94) between OCC pickup rate and temperature. The OCC pickup rate assay can be used experimentally, and should be valuable in evaluating factors that affect rate and in studies dealing with the mechanism of OCC pickup. Mol. Reprod. Dev. 47:312–322, 1997. © 1997 Wiley-Liss, Inc.     

The evolution of the life cycle of brown seaweeds

The brown seaweeds (Phaeophyta) are well-suited for testing theories of the evolution of the sexual alternation of haploid and diploid generations because of the great diversity of life cycles within the phylum. Three theories are investigated in this paper. (1) Diploid growth evolves because it has the effect of complementing deleterious recessive mutations. This is rejected because (a) ancestral haplonty is not a parsimonious inference from current phylogenies; (b) the exaggeration of diploid growth does not evolve in a comb-like fashion; (c) forms with predominantly haploid growth have evolved from smaller isomorphic ancestors; and (d) there is no correlation between haploid growth and monoecy. (2) Diploid growth evolves when gamete dimorphism leads to intense sexual selection, favouring the production of genetically diverse gametes through meiosis. This is rejected because diere is no correlation between the dominance of the diploid generation and the degree of gamete dimorphism. It is possible to show that gamete dimorphism itself has evolved in the Phaeophyta through the increase in size of the macrogamete in forms that have evolved larger sporophytes. (3) Microthalli become specialized as gametophytes because fusion is promoted by releasing gametes into the boundary layer; macrothalli become specialized as sporophytes because dispersal is promoted by releasing zoospores into the water column. This is consistent with the sexual and reproductive biology of Phaeophyta. The classic sexual cycle can then be interpreted as evolving from an asexual alternation of microthallus and macrothallus, governed largely by environmental factors, through selection for the appropriate association of ploidy with vegetative size. The exceptions to this general rule are forms in which gametes are released from macrothalli, where a different suite of sexual characters has evolved.     

Autophagy is required for gamete differentiation in the moss Physcomitrella patens

Autophagy, a major catabolic process in eukaryotes, was initially related to cell tolerance to nutrient depletion. In plants autophagy has also been widely related to tolerance to biotic and abiotic stresses (through the induction or repression of programmed cell death, PCD) as well as to promotion of developmentally regulated PCD, starch degradation or caloric restriction important for life span. Much less is known regarding its role in plant cell differentiation. Here we show that macroautophagy, the autophagy pathway driven by engulfment of cytoplasmic components by autophagosomes and its subsequent degradation in vacuoles, is highly active during germ cell differentiation in the early diverging land plant Physcomitrella patens. Our data provide evidence that suppression of ATG5-mediated autophagy results in reduced density of the egg cell-mediated mucilage that surrounds the mature egg, pointing toward a potential role of autophagy in extracellular mucilage formation. In addition, we found that ATG5- and ATG7-mediated autophagy is essential for the differentiation and cytoplasmic reduction of the flagellated motile sperm and hence for sperm fertility. The similarities between the need of macroautophagy for sperm differentiation in moss and mouse are striking, strongly pointing toward an ancestral function of autophagy not only as a protector against nutrient stress, but also in gamete differentiation.     

Chemically moderated gamete preferences predict offspring fitness in a broadcast spawning invertebrate

Sperm chemoattraction, where sperm locate unfertilized eggs by following a concentration gradient of egg-derived chemoattractants, has been widely documented across numerous taxa. While marine invertebrates are favoured models for understanding the underlying mechanisms of sperm chemoattraction, the evolutionary forces underpinning the process remain enigmatic. Here, we show that in mussels (Mytilus galloprovincialis), chemically moderated gamete preferences promote assortative fertilizations between genetically compatible gametes. When offered the choice of egg clutches from two females, sperm exhibited consistent but differential ‘preferences’ for chemical cues secreted from conspecific eggs. Critically, our data reveal that the preferences shown by sperm during the egg-choice trials are highly predictive of early embryonic viability when eggs and sperm from the same individuals are mixed during standard (no-choice) fertilization assays. Moreover, we demonstrate that by experimentally separating chemoattractants from eggs, sperm swimming behaviour is differentially regulated by egg-derived chemoattractants, and that these changes in sperm behaviour are highly consistent with observed patterns of gamete preferences, fertilization and larval survival. Together, this integrated series of experiments reveals that the behaviour of sperm is fine-tuned to respond differentially to the chemical signals emitted from different conspecific eggs, and that these choices have measurable fitness benefits.     

'土佐文旦'胚囊发育及其子代体细胞染色体的观察研究

选取母本‘土佐文旦’不同时期的花蕾及文旦杂交F1子代3~5 mm长的幼叶,采用石蜡制片法及柑橘染色体制片技术,观察不同时期母本子房结构及杂交F1子代体细胞染色体数目。结果显示:(1)追踪到母本大孢子的发生及不同时期胚囊的发育特征,获得了杂交F1子代清晰可靠、分散良好的体细胞染色体图像,母本石蜡切片观察结果为进一步确定2n雌配子的最佳诱导期提供依据;(2)杂交F1子代体细胞染色体观察结果表明,杂交F1子代中既有正常的二倍体也有三倍体,为进一步原位杂交技术及确认2n配子类型和可能的遗传效应奠定了基础。     

DETECTION AND EVALUATION OF A NOVEL SEXUAL PHEROMONE THAT INDUCES SEXUAL CELL DMSION OF CLOSTERIUM EHRENBERGII (CHLOROPHYTA)1

Mating type-plus (mt⁺; NIES-228) cells of Closterium ehrenbergii undergo a division to form gamete-shaped cells. This cell division is induced by a substance produced by mating type-minus (mt^?; NIES-229) cells. Light and the presence of mt⁺ cells enhanced production of the substance. The active substance is heat labile and has an apparent molecular mass of 20 kDa. From these results, we conclude that the substance is a novel, proteinaceous sexual pheromone involved in reproduction of Closterium ehrenbergii.     

Protein-carbohydrate complementarity in mammalian gamete recognition

Recent studies suggest that gamete recognition in a number of species is mediated by complementary proteins and carbohydrates on opposing gamete surfaces. Studies in invertebrates and vertebrates have shown that carbohydrate-binding proteins on the sperm surface recognize and bind to complementary glycoconjugates on the egg's extracellular coat. This chapter reviews our current knowledge of gamete recognition in the mouse. The complementary receptors for both mouse sperm and egg have been identified, purified, and characterized. Their synthesis during gametogenesis has been defined, as have the effects of sperm capacitation and of the acrosome reaction on their expression and distribution. Their relationship to gamete receptors that function in other species is discussed. Finally, evidence is presented that suggests that one of the receptors that mediate mouse gamete recognition belongs to a family of cell surface receptors that function during multiple cellular interactions in development.     

Interaction of isolated components from mammalian sperm and egg

In order to identify those proteins from the plasma membrane of hamster spermatozoa that exhibit an affinity for components of the zona pellucida we have used the Western blot technique. Zonae pellucidae from postovulatory hamster oocytes were solubilized by exposure to an acidic pH and then radiolabeled using the Bolton-Hunter reagent. These ¹²⁵I-zona pellucida proteins retain their immunoreactivity and migrate in three heterogeneous bands when submitted to SDS-PAGE electrophoresis. Membrane proteins from epididymal spermatozoa of mature hamsters were extracted by treatment with Nonidet P-40 and then submitted to electrophoresis (SDS-PAGE). The proteins in the gel were electrophoretically transferred to a nitrocellulose membrane and then probed with the radiolabelled zone pellucida proteins. ¹²⁵I-zona pellucida proteins bind preferentially to a sperm protein with a molecular weight of 26,400 ± 1,400 daltons (n = 9). Using a similar procedure it was shown that this protein also binds ¹²⁵I-Concanavalin A. The interaction between the sperm protein and the ¹²⁵I-zona pellucida proteins shows species specificity as demonstrated by the fact that the hamster ¹²⁵I-zona pellucida proteins do not bind to proteins extracted from ram, bull, and stallion spermatozoa. Whether this sperm protein could be implicated be implicated in the process of sperm-egg interaction is under investigation.     

The Caenorhabditis elegans spe‐49 gene is required for fertilization and encodes a sperm‐specific transmembrane protein homologous to SPE‐42

Fertilization, the fusion of sperm and oocyte to form a zygote, is the first and arguably the most important cell–cell interaction event in an organism’s life. Forward and reverse genetic approaches in the nematode Caenorhabditis elegans have identified many genes that are required for gametogenesis and fertilization and thus are beginning to elucidate the molecular pathways that underlie these processes. We identified an allele of the spe‐49 gene in a second filial generation (F₂) mutagenesis screen for spermatogenesis‐defective (spe) mutants. Mutant worms for spe‐49 produce sperm that have normal morphology, activate to form ameboid spermatozoa, and can migrate to and maintain their position in the hermaphrodite reproductive tract but fail to fertilize oocytes. This phenotype puts spe‐49 in the spe‐9 class of late‐acting genes that function in sperm at the time of fertilization. We cloned the spe‐49 gene through a combination of deficiency mapping, transgenic rescue, and genomic sequencing. spe‐49 messenger RNA (mRNA) is enriched in male germ cells, and the complementary DNA (cDNA) encodes a predicted 772‐amino‐acid six‐pass transmembrane protein that is homologous to SPE‐42. Indeed, SPE‐49 and SPE‐42 have identical predicted membrane topology and domain structure, including a large extracellular domain with six conserved cysteine residues, a DC‐STAMP domain, and a C‐terminal cytoplasmic domain containing a C4–C4 RING finger motif. The presence of two SPE‐42 homologs in animal genomes from worms to humans suggests that these proteins are highly conserved components of the molecular apparatus required for the sperm–oocyte recognition, binding, and fusion.