All males are not created equal: Fertility differences depend on gamete recognition polymorphisms in sea urchins

Behaviors, morphologies, and genetic loci directly involved in reproduction have been increasingly shown to be polymorphic within populations. Explaining how such variants are maintained by selection is crucial to understanding the genetic basis of fertility differences, but direct tests of how alleles at reproductive loci affect fertility are rare. In the sea urchin genus Echinometra, the protein bindin mediates sperm attachment to eggs, evolves quickly, and is polymorphic within species. Eggs exposed to experimental sperm mixtures show strong discrimination on the basis of the males’ bindin genotype. Different females produce eggs that nonrandomly select sperm from different males, showing that variable egg–sperm interactions determine fertility. Eggs select sperm with a bindin genotype similar to their own, suggesting strong linkage between female choice and male trait loci. These experiments demonstrate that alleles at a single locus can have a strong effect on fertilization and that reproductive loci may retain functional polymorphisms through epistatic interactions between male and female traits. They also suggest that positive selection at gamete recognition loci like bindin involves strong selection within species on mate choice interactions.

A Model for Signal Transduction during Gamete Release in the Fucoid Alga Pelvetia compressa1

Fucoid algae release gametes into seawater following an inductive light period (potentiation), and gamete expulsion from potentiated receptacles of Pelvetia compressa began about 2 min after a light-to-dark transition. Agitation of the medium reversed potentiation, with an exponential time course completed in about 3 h. Light regulated two signaling pathways during potentiation and gamete expulsion: a photosynthetic pathway and a photosynthesis-independent pathway in which red light was active but blue light was not. Uptake of K+ appears to have an important role in potentiation, because a 50% inhibition of potentiation occurred in the presence of the tetraethylammonium ion, a K+-channel blocker. A central role of anion channels in the maintenance of potentiation is suggested by the premature release of gametes in the light when receptacles were incubated with inhibitors of slow-type anion channels. An inhibitor of tyrosine kinases, tyrphostin A63, also inhibited potentiation. A model for gamete release from P. compressa is presented that proposes that illumination results in the accumulation of ions (e.g. K+) throughout the cells of the receptacle during potentiation, which then move into the extracellular matrix during gamete expulsion to generate osmomechanical force, resulting in gamete release.

Stimulation of renin secretion by nitric oxide is mediated by phosphodiesterase 3

This study aimed to characterize the cellular pathways along which nitric oxide (NO) stimulates renin secretion from the kidney. Using the isolated perfused rat kidney model we found that renin secretion stimulated 4- to 8-fold by low perfusion pressure (40 mmHg), by macula densa inhibition (100 μmol/liter of bumetanide), and by adenylate cyclase activation (3 nmol/liter of isoproterenol) was markedly attenuated by the NO synthase inhibitor nitro-l-arginine methyl ester (l-Name) (1 mM) and that the inhibition by l-Name was compensated by the NO-donor sodium nitroprusside (SNP) (10 μmol/liter). Similarly, inhibition of cAMP degradation by blockade of phosphodiesterase 1 (PDE-1) (20 μmol/liter of 8-methoxymethyl-1-methyl-3-(2-methylpropyl)xanthine) or of PDE-4 (20 μmol/liter of rolipram) caused a 3- to 4-fold stimulation of renin secretion that was attenuated by l-Name and that was even overcompensated by sodium nitroprusside. Inhibition of PDE-3 by 20 μmol/liter of milrinone or by 200 nmol/liter of trequinsin caused a 5- to 6-fold stimulation of renin secretion that was slightly enhanced by NO synthase inhibition and moderately attenuated by NO donation. Because PDE-3 is a cGMP-inhibited cAMP-PDE the role of endogenous cGMP for the effects of NO was examined by the use of the specific guanylate cyclase inhibitor 1-H-(1,2,4)oxodiazolo(4,3a)quinoxalin-1-one (20 μmol). In the presence of 1H-[1,2,4]oxodiazolo[4,3-a]quinoxalin-1-one the effect of NO on renin secretion was abolished, whereas PDE-3 inhibitors exerted their normal effects. These findings suggest that PDE-3 plays a major role for the cAMP control of renin secretion. Our findings are compatible with the idea that the stimulatory effects of endogenous and exogenous NO on renin secretion are mediated by a cGMP-induced inhibition of cAMP degradation.

Rapid evolution of sex-related genes in Chlamydomonas

Biological speciation ultimately results in prezygotic isolation—the inability of incipient species to mate with one another–but little is understood about the selection pressures and genetic changes that generate this outcome. The genus Chlamydomonas comprises numerous species of unicellular green algae, including numerous geographic isolates of the species C. reinhardtii. This diverse collection has allowed us to analyze the evolution of two sex-related genes: the mid gene of C. reinhardtii, which determines whether a gamete is mating-type plus or minus, and the fus1 gene, which dictates a cell surface glycoprotein utilized by C. reinhardtii plus gametes to recognize minus gametes. Low stringency Southern analyses failed to detect any fus1 homologs in other Chlamydomonas species and detected only one mid homolog, documenting that both genes have diverged extensively during the evolution of the lineage. The one mid homolog was found in C. incerta, the species in culture that is most closely related to C. reinhardtii. Its mid gene carries numerous nonsynonymous and synonymous codon changes compared with the C. reinhardtii mid gene. In contrast, very high sequence conservation of both the mid and fus1 sequences is found in natural isolates of C. reinhardtii, indicating that the genes are not free to drift within a species but do diverge dramatically between species. Striking divergence of sex determination and mate recognition genes also has been encountered in a number of other eukaryotic phyla, suggesting that unique, and as yet unidentified, selection pressures act on these classes of genes during the speciation process.

ApoNifH functions in iron–molybdenum cofactor synthesis and apodinitrogenase maturation

NifH (dinitrogenase reductase) has three important roles in the nitrogenase enzyme system. In addition to its role as the obligate electron donor to dinitrogenase, NifH is required for the iron–molybdenum cofactor (FeMo-co) synthesis and apodinitrogenase maturation. We have investigated the requirement of the Fe–S cluster of NifH for these processes by preparing apoNifH. The 4Fe–4S cluster of NifH was removed by chelation of the cluster with α, α′-bipyridyl. The resulting apoNifH was tested in in vitro FeMo-co synthesis and apodinitrogenase maturation reactions and was found to function in both these processes. Thus, the presence of a redox active 4Fe–4S cluster in NifH is not required for its function in FeMo-co synthesis and in apodinitrogenase maturation. This, in turn, implies that the role of NifH in these processes is not one of electron transfer or of iron or sulfur donation.

Xenopus Cdc6 confers sperm binding competence to oocytes without inducing their maturation

Amphibian eggs normally require meiotic maturation to be competent for fertilization. A necessary prerequisite for this event is sperm binding, and we show that under normal physiological conditions this property is acquired at, but not before, meiotic maturation. Immature oocytes do not bind sperm, but injection of total egg poly(A)+ mRNA into immature oocytes confers sperm binding in the absence of meiotic maturation. Using an expression cloning approach we have isolated a single cDNA from egg poly(A)+ mRNA that can induce sperm binding in immature oocytes. The cDNA was found to encode Xenopus Cdc6, a protein that previously has been shown to function in initiation of DNA replication and cell cycle control. This unanticipated finding provides evidence of a link between a regulator of the cell cycle and alterations in cell surface properties that affect gamete binding.

Activation of mouse sperm T-type Ca2+ channels by adhesion to the egg zona pellucida

The sperm acrosome reaction is a Ca2+-dependent exocytotic event that is triggered by adhesion to the mammalian egg’s zona pellucida. Previous studies using ion-selective fluorescent probes suggested a role of voltage-sensitive Ca2+ channels in acrosome reactions. Here, whole-cell patch clamp techniques are used to demonstrate the expression of functional T-type Ca2+ channels during mouse spermatogenesis. The germ cell T current is inhibited by antagonists of T-type channels (pimozide and amiloride) as well as by antagonists whose major site of action is the somatic cell L-type Ca2+ channel (1,4-dihydropyridines, arylalkylamines, benzothiazapines), as has also been reported for certain somatic cell T currents. In sperm, inhibition of T channels during gamete interaction inhibits zona pellucida-dependent Ca2+ elevations, as demonstrated by ion-selective fluorescent probes, and also inhibits acrosome reactions. These studies directly link sperm T-type Ca2+ channels to fertilization. In addition, the kinetics of channel inhibition by 1,4-dihydropyridines suggests a mechanism for the reported contraceptive effects of those compounds in human males.

Male gametic cell-specific gene expression in flowering plants

The role of the male gamete—the sperm cell—in the process of fertilization is to recognize, adhere to, and fuse with the female gamete. These highly specialized functions are expected to be controlled by activation of a unique set of genes. However, male gametic cells traditionally have been regarded as transcriptionally quiescent because of highly condensed chromatin and a very reduced amount of cytoplasm. Here, we provide evidence for male gamete-specific gene expression in flowering plants. We identified and characterized a gene, LGC1, which was shown to be expressed exclusively in the male gametic cells. The gene product of LGC1 was localized at the surface of male gametic cells, suggesting a possible role in sperm–egg interactions. These findings represent an important step toward defining the molecular mechanisms of male gamete development and the cellular processes involved in fertilization of flowering plants.

A calcium influx is triggered and propagates in the zygote as a wavefront during in vitro fertilization of flowering plants

In this paper, we report direct measurement of an influx of extracellular Ca2+ induced by gamete fusion in flowering plants. This result was obtained during maize in vitro fertilization with the use of an extracellular Ca2+-selective vibrating probe. Ca2+ influx recorded at the surface of isolated egg cells, with or without adhesion of a male sperm cell, was close to zero and stable over time. Gamete fusion, however, triggered a Ca2+ influx in the vicinity of the sperm entry site with a delay of 1.8 ± 0.6 sec. The Ca2+ influx spread subsequently through the whole egg cell plasma membrane as a wavefront, progressing at an estimated rate of 1.13 μm⋅sec−1. Once established, Ca2+ influx intensities were sustained, monotonic and homogeneous over the whole egg cell, with an average peak influx of 14.92 pmol⋅cm−2⋅sec−1 and an average duration of 24.4 min. The wavefront spread of channel activation correlates well with the cytological modifications induced by fertilization, such as egg cell contraction, and with the cytosolic Ca2+ (c[Ca2+]) elevation previously reported. Calcium influx was inhibited effectively by gadolinium, possibly implicating mechanosensitive channels. Furthermore, artificial influxes created by incubation with Ca2+ ionophores mimicked some aspects of egg activation. Taken together, these results suggest that, during fertilization in higher plants, gamete membrane fusion starts the first embryonic events by channel opening and Ca2+ influx. In turn, c[Ca2+] may work as a trigger and possibly a space and time coordinator of many aspects of egg activation.

Successful external fertilization in turbulent environments.

Mathematical and experimental simulations predict that external fertilization is unsuccessful in habitats characterized by high water motion. A key assumption of such predictions is that gametes are released in hydrodynamic regimes that quickly dilute gametes. We used fucoid seaweeds to examine whether marine organisms in intertidal and subtidal habitats might achieve high levels of fertilization by restricting their release of gametes to calm intervals. Fucus vesiculosus L. (Baltic Sea) released high numbers of gametes only when maximal water velocities were below ca. 0.2 m/s immediately prior to natural periods of release, which occur in early evening in association with lunar cues. Natural fertilization success measured at two sites was always close to 100%. Laboratory experiments confirmed that (i) high water motion inhibits gamete release by F. vesiculosus and by the intertidal fucoids Fucus distichus L. (Maine) and Pelvetia fastigiata (J. Ag.) DeToni (California), and (ii) showed that photosynthesis is required for high gamete release. These data suggest that chemical changes in the boundary layer surrounding adults during photosynthesis and/or mechanosensitive channels may modulate gamete release in response to changing hydrodynamic conditions. Therefore, sensitivity to environmental factors can lead to successful external fertilization, even for species living in turbulent habitats.

Production of transgenic dwarf surfclams, Mulinia lateralis, with pantropic retroviral vectors.

A pantropic pseudotyped retroviral vector containing the envelope protein of vesicular stomatitis virus was used as a gene transfer vector in the dwarf surfclam, Mulinia lateralis. These pantropic retroviral vectors have an extremely broad host cell range and can infect many nonmammalian species. Newly fertilized dwarf surfclam eggs were electroporated at 700 V in the presence of 1 x 10(4) colony-forming units of pantropic pseudotyped retroviral particles. Infection was well tolerated and did not affect the survival rate of the embryos. Gametes collected from P1 presumptive transgenic animals were analyzed for the presence of provirus by PCR, and in different experiments 13-33% of the gamete pools were positive for the transgene. Dot blot hybridization of DNA samples from the F1 offspring of two different crosses between infected P1 and wild-type individuals revealed that 28% and 31% of F1 offspring were transgenic, respectively. Southern blot analysis of DNA isolated from PCR-positive F1 animals confirmed integration of a single copy of the provirus into the host genome. Thus, the germ lines of these two P1 transgenic animals were mosaic for the transgene. Expression of beta-galactosidase encoded by the provirus was detected in transgenic but not control surfclam embryos. Pantropic pseudotyped retroviral vectors provide a useful method for the stable introduction of foreign genetic information into surfclams and may facilitate the introduction of desirable genetic traits into commercially important shellfish and crustaceans.

Extraordinary divergence and positive Darwinian selection in a fusagenic protein coating the acrosomal process of abalone spermatozoa.

During fertilization in marine invertebrates, fusion between sperm and egg cell membranes occurs at the tip of the sperm acrosomal process. In abalone sperm the acrosomal process is coated with an 18-kDa protein. In situ, this protein has no effect on the egg vitelline envelope, but in vitro it is a potent fusagen of liposomes. Thus, the 18-kDa protein may mediate membrane fusion between the gametes, a step in gamete recognition known to restrict heterospecific fertilization in other species. The cDNA and deduced amino acid sequences of the 18-kDa protein were determined for five species of California abalone. The deduced amino acid sequences exhibit extraordinary divergence; the percent identity varies from 27% to 87%. Analysis of nucleotide substitution shows extremely high frequencies of amino acid-altering substitution compared to silent substitution, demonstrating that positive Darwinian selection promotes the divergence of this protein. However, amino acid replacement is conservative with respect to size and polarity of residue. The data support the developing idea that in free-spawning marine invertebrates, the proteins mediating fertilization may be subjected to intense, and as yet unknown, selective forces. The extraordinary divergence of fertilization proteins may be related to the establishment of barriers to heterospecific fertilization.