Analysis of a chalcone synthase mutant in Ipomoea purpurea reveals a novel function for flavonoids: amelioration of heat stress

Flavonoids are thought to function in the plant stress response and male fertility in some, but not all, species. We examined the effects of a self-fertile chalcone synthase null allele, a, for the effects of heat and light stress on fertilization success and flower production in Ipomoea purpurea. Pollen recipients and pollen donors of both homozygous genotypes exhibit reduced fertilization success at high temperatures, indicating that high temperature acts as a stress-lowering fertilization success. Homozygous aa individuals exhibit reduced male and female fertilization success, compared to AA individuals, at high temperatures but not at low temperatures. In addition, aa individuals produce fewer flowers than AA individuals at low temperatures, but not at high temperatures. These results suggest that flavonoids alleviate heat stress on fertilization success. They also suggest that pleiotropic effects at the A locus may explain the low frequency of the a allele in natural populations.     

Immunoglobulins against gp273, the ligand for sperm-egg interaction in the mollusc bivalve Unio elongatulus,are directed against charged O-linked oligosaccharide chains bearing a Lewis-like structure and interact with epitopes of the human zona pellucida

In oocytes of the mollusc bivalve Unio elongatulus, gp273 is the ligand molecule for sperm-egg interaction and binding is mediated by its O-glycans. A serum raised against this protein enabled its localization in the crater region, the area of the vitelline coat where sperm recognition occurs, and showed that after cyanogen bromide fragmentation, the anti-gp273 epitope(s) was retained by a peptide where the O-glycans are localized. In this article, we utilized purified anti-gp273 immunoglobulins to characterize the corresponding epitope by: (i) immunoblotting analysis of the protein after removal of O- and N-glycans; (ii) solid phase binding analysis of anti-gp273 IgG to gp273 N- and O-glycans; and (iii) binding analysis of the same antibody to commercially available oligosaccharides. The results showed that the epitope consists of O-glycans and contains a Lewis-like structure with fucose as determinant. Anti-gp273 IgG were then used to investigate human zona pellucida by immunoelectronmicroscopy and immunoblotting. Epitopes recognized by the antibody were demonstrated on the outer surface of the zona pellucida and shown to belong to a zona pellucida protein having electrophoretic mobility similar to human ZP3. Since human sperm specifically bind to gp273, and anti-gp273 interferes with this binding a functional role for these epitopes is suggested.     

Exocytosis of a 60 kDa protein (calreticulin) from activated hamster oocytes

The sp50 protein localized at the acrosomal region of guinea pig sperm was suggested to participate in acrosome exocytosis, the acrosome reaction (AR). On the other hand, the cortical reaction (CR), also an exocytotic event, occurs during egg activation. The aim of the present work was to identify sp50 and also to define if sp50 is present in hamster eggs, as well as its location before and after CR. Sp50 was identified as calreticulin (CRT), based on: (a) its NH(2)-terminal amino acid (25 aa) sequence, (b) a cross-recognition of pure sp50 and pure CRT with anti-CRT (from Santa Cruz, anti-CRTsc), and anti-sp50 (anti-sp50/CRT) antibodies, respectively, and (c) that both antibodies revealed a 50 kDa protein in a Brij sperm extract. On the other hand, CRT presence in eggs was positively determined by Western blotting (Wb) using anti-sp50/CRT antibody which recognized a 60 kDa protein in the egg extract, and by indirect immunofluorescence (IIF), CRT was located in the cortical granules (CG). It was defined by a granular pattern and co-localization with mannose, a specific carbohydrate of the CG. Additionally, a decrease in CRT concentration occurred in eggs after their activation and, in parallel, the protein was revealed in the egg's incubation medium. In activated eggs with zona pellucida (ZP), CRT remains as a halo in the perivitelline space and around the polar body. From these results we suggest that: (1) CRT is present in the CG of non-activated hamster eggs, (2) CRT is exocytosed during the CR, in response to egg activation, and (3) CRT might participate in the block to polyspermy, together with other CG components.     

Essential role of the nonreducing terminal alpha-mannosyl residues of the N-linked carbohydrate chain of bovine zona pellucida glycoproteins in sperm-egg binding

It has been proposed that mammalian sperm bind species-specifically to carbohydrate chains of zona pellucida glycoproteins at fertilization. Although the sperm ligand carbohydrate chains have been characterized in mice and pigs, the existence of the ligands of other mammals remains unclear. In order to explore the bovine sperm ligand, two in vitro competition assay methods were applied. As a result, a high-mannose-type carbohydrate chain, Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-3)Manbeta1-4GlcNAcbeta1-4GlcNAc, which is the major neutral chain in bovine egg zona glycoproteins, was shown to possess bovine sperm ligand activity. When nonreducing terminal alpha-mannosyl residues were eliminated from the zona glycoproteins by alpha-mannosidase digestion, the ligand activity was reduced, indicating that the alpha-mannosyl residues play an essential role in bovine sperm-egg binding. The number of sperm binding to eggs was reduced to about one-half after fertilization. The ligand-active high-mannose-type chain may be buried after fertilization, since its amount remains unchanged. Pretreatment of bovine sperm with the sperm ligand-carbohydrate chain significantly inhibited penetration of the sperm into oocyte and the male pronucleus formation. Thus, a correlation between the sperm ligand activity and in vitro fertilization rate was observed.     

Microsatellite identification of extrapair sires in a socially monogamous warbler

Few studies of avian mating systems have identified the siresof extrapair young, and hence it has been difficult to determinethe scale at which reproductive interactions occur. For instance,females may be free to copulate with any male in the population(a "global" scale of interactions), or females may be restrictedto copulating only with males on neighboring territories (a"local" scale). The scale of such interactions has importantconsequences for an understanding of the evolutionary causesand consequences of extrapair fertilizations. We used five hypervariable microsatellite loci and multilocus DNA fingerprintingto examine parentage of more than 400 nestling black-throatedblue warblers (Dendroica caerulescens). Extrapair fertilizationswere common, and the microsatellite markers allowed us to identifythe sires for 89% of the young analyzed. Most identified extrapairsires were males on neighboring or nearby territories, andmost nestlings for whom we could not identify a sire came fromterritories at the edge of the study plot. Thus, reproductive interactions appear to be more local than global in this population.Extrapair fertilizations contributed significantly to totalvariation in male reproductive success. However, the standardizedvariance in male reproductive success (0.68-0.74) was not substantiallygreater than that for females (0.53-0.60), and the contributionof extrapair fertilizations (9-14%) was much lower than thecontribution of within-pair fertilizations (75-77%). This suggeststhat the local scale of reproductive interactions may limitvariation in male reproductive success and hence the opportunityfor selection.     

Acid glycohydrolases in rat spermatocytes, spermatids and spermatozoa: enzyme activities, biosynthesis and immunolocalization

Mammalian sperm acrosomes contain several glycohydrolases that are thought to aid in the dispersion and digestion of vestments surrounding the egg. In this study, we have used multiple approaches to examine the origin of acrosome-associated glycohdyrdolases. Mixed spermatogenic cells, prepared from rat testis, were separated by unit gravity sedimentation. The purified germ cells (spermatocytes [SC], round spermatids [RS], and elongated/condensed spermatids [E/CS]) contained several glycohydrolase activities. Metabolic labeling in the cell culture, immunoprecipitation, and autoradiographic approaches revealed that β-D-galactosidase was synthesized in SC and RS in 88/90 kDa forms which undergo processing in a cell-specific manner. Immunohistochemical approaches demonstrated that the enzyme was localized in Golgi membranes/vesicles, and lysosome-like structures in SC and RS, and forming/formed acrosome of E/CS. Published December 3, 2001     

Electron microscopic observations of karyogamy in the fish egg

In the initial step of pronuclear association in fertilized fish eggs, the female and male pronuclei (containing large nucleolus-like bodies) were juxtaposed in the center of the blastodisc and formed nucleoplasmic projections along adjacent surfaces. After contact of the pronuclei, small internuclear bridges joining them were formed by fusion at several regions of the nuclear envelope projections. No specific site of fusion or breakdown of nuclear envelopes was recognized in the pronuclei during karyogamy. In the advanced stage, clumps of condensing chromatin appeared in the nucleoplasm of the newly fused pronuclei. The number and diameter of the internuclear bridges increased gradually by progressive fusion in many regions, finally yielding a spherical zygote nucleus. Following complete formation of the zygote nucleus, the pronuclear envelope began to break down concomitantly with shrinkage of the nucleoplasm, which was highly convoluted around the entire nuclear surface. The nucleoplasm containing chromosomes then mingled with the perinuclear cytoplasm.     

Egg-to-embryo transition is driven by differential responses to Ca(2+) oscillation number

Ca(2+) oscillations and signaling represent a basic mechanism for controlling many cellular events. Activation of mouse eggs entrains a temporal series of Ca(2+)-dependent events that include cortical granule exocytosis, cell cycle resumption with concomitant decreases in MPF and MAP kinase activities, and recruitment of maternal mRNAs. The outcome is a switch in cellular differentiation, i.e., the conversion of the egg into the zygote. By activating mouse eggs with experimentally controlled and precisely defined Ca(2+) transients, we demonstrate that each of these events is initiated by a different number of Ca(2+) transients, while their completion requires a greater number of Ca(2+) transients than for their initiation. This combination of differential responses to the number of Ca(2+) transients provides strong evidence that a single Ca(2+) transient-driven signaling system can initiate and drive a cell into a new developmental pathway, as well as can account for the temporal sequence of cellular changes associated with early development.     

Precambrian animal life: probable developmental and adult cnidarian forms from Southwest China

We have previously described the affinity of a pig sperm surface protein, P68, to mammalian zonae pellucidae (ZP). In this report, we identified P68 as arylsulfatase A (AS-A) based on the presence of P68 tryptic peptide sequences in the pig testis AS-A cDNA sequence. Our objective was to demonstrate the presence of AS-A on the sperm surface and to elucidate its role in ZP binding. Immunogold electron microscopy revealed the presence of AS-A on the sperm surface. Furthermore, live pig sperm and the extract of peripheral sperm plasma membrane proteins exhibited AS-A's desulfation activity. Significantly, the role of pig sperm surface AS-A in ZP binding was demonstrated by dose-dependent decreases of sperm-ZP binding upon sperm pretreatment with anti-AS-A IgG/Fab, and by the binding of Alexa-430-conjugated sperm surface AS-A to homologous ZP. ZP pretreatment with anti-pig-ZP3 antibody abolished AS-A binding, suggesting that ZP3, recognized as the pig sperm receptor, was AS-A's binding ligand. This was further confirmed by the ability of exogenous ZP3 to competitively inhibit AS-A-ZP binding. Similarly, purified ZP3alpha, a major sperm receptor component of ZP3, exhibited great inhibitory effect on AS-A-ZP binding. All of these results designated a new function of AS-A in gamete interaction.     

The synergid cell: attractor and acceptor of the pollen tube for double fertilization

In flowering plants, the egg cell is generally accompanied by two symmetrical cells, called synergid cells. As early as the 1870s, synergid cells were distinguished from egg cells and cooperation between synergid and egg cells was proposed; the term "synergid" is derived from the Greek "synergos," which means "working together." The accumulation of morphological and genetic data, and, more recently, the in vitro physiological analysis of the fertilization system of Torenia fournieri, have revealed that synergid cells work together with egg and central cells to accomplish double fertilization. This cooperation is of crucial importance in the attraction and acceptance of the pollen tube. In this review article, I focus on the physiological function and behavior of the synergid cell during the fertilization process. Received: December 20, 2001 / Accepted: December 27, 2001