Individual Gametes Research Articles

Conversion of Fertility Restoration of the Sorghum IS1112C (A3) Male‐Sterile Cytoplasm from Two Genes to One Gene

The restoration of male fertility in sorghum [Sorghum bicolor (L.) Moench] lines carrying the IS1112C male‐sterile cytoplasm is characterized by limited seed set. Restoration requires complementary gametophytic action of two restoring alleles, Rf3 and Rf4, for individual gamete viability, and an F1 heterozygous for the two restoring loci is expected to exhibit 25% viable pollen. The objective of this study was to demonstrate the feasibility of converting restoration to single gene behavior. Single‐seed descent B3Tx398/IS1112C F5:6 lines were used to generate homozygosity at the rf3 and rf4 loci, and critical segregants were identified by progeny tests, assays for action of the Rf3 allele, and genomic markers for the rf4 locus. Using these criteria, we constructed the genotypes Rf3Rf3rf4rf4 and rf3rf3Rf4Rf4 in normal, male‐fertile cytoplasm lines, and in IS1112C male‐sterile cytoplasm lines. Pollination of either male‐sterile line with a matching male‐fertile line resulted in about 25% pollen staining in the F1, demonstrating complementation of the two restoring loci. Pollination of either male‐sterile line with IS1112C, Rf3Rf3Rf4Rf4, resulted in about 50% pollen staining. These characteristics substantiate complementary action of the restoring alleles, and are consistent with successful conversion to a single gene fertility restoration system.

Sex and Selection: a Reply to Matthen

Mohan Matthen ([1999]) argues that when reproduction is sexual, natural selection can explain why individual organisms possess the traits they do. In stating his argument Matthen makes use of a conception of individual organisms as receptacles for collections of genes—a conception that cannot do the work Matthen requires of it. Either these receptacles are abstract objects, such as bare possibilities for organisms, or they are concrete. The first reading is too weak, since it allows selection to explain individual traits in both sexual and asexual contexts. The only concrete entities we might think of as receptacles for collections of genes are male or female gametes. It is true that in the sexual context selection explains why an individual gamete combines with a second gamete of one type rather than another; however, this is not to say that selection explains why an individual organism has the traits it does.

Sperm precedence in a novel context: mating in a sessile marine invertebrate with dispersing sperm.

The compound ascidian Diplosoma listerianum releases aquatic sperm which are dispersed passively to potential mates as individual gametes prior to storage of sperm, internal fertilization and brooding of embryos. The storage of exogenous sperm enables D. listerianum to produce a lengthy series of progeny following a brief period of mating. Molecular paternity analysis following sequential mating of colonies in laboratory culture revealed a consistent pattern with a clear initial bias in paternity towards the first of two acting males. The sites of sperm storage and fertilization and the morphology of the ovary in D. listerianum suggest that this bias reflects first-in-first-out use of individual stored gametes. The proportion of second-male paternity subsequently increased with time within the progeny arrays. This may have reflected the ageing or passive loss of first-male sperm. It is also possible that the modular nature of the organism contributed to this temporal trend: any recently budded colony modules maturing in the interval between matings would have been available exclusively to second-male sperm as virgin zooids. Two sets of mating trials were run. In the first, the collection of progeny suffered an interruption of 13 days and each male gained a larger proportion of recorded paternity within the progeny analysed when mating first rather than when mating second. In one mating combination, the first male obtained almost 100% of recorded paternity. In the second set of trials, with different clonal combinations, the complete sequence of progeny was collected and the estimated overall proportion of second-male paternity (P2) was consistently > 0.5. Taken as a whole, the results suggest that the overall P2-value can vary widely within the population studied. Proposed mechanisms of mating-order effects in species with copulatory mating include several which can have no counterpart in indirect aquatic mating since they involve the active removal, sealing off, volumetric displacement or incapacitation of first-male ejaculates. It is nevertheless clear that mating-order effects can be pronounced during the type of non-copulatory mating examined here, which is widespread in marine invertebrates.

Density-related reproductive trade-offs in the green sea urchin, Strongylocentrotus droebachiensis

Green sea urchin, Strongylocentrotus droebachiensis (O.F. Muller), populations are being depleted rapidly in the Gulf of Maine and there is justified concern that the potential of this free-spawner to produce larvae may be severely inhibited. We evaluated the opposing effects of different population densities on gonad development and fertilization success, using population surveys and fertilization experiments. We determined gonad indices (gonad mass/body mass) over a range of population densities (0.1 to 250 ind. m−2) at seven sites in coastal Maine, USA, sampled at two depths (5 and 15 m). At shallow sites, we found that gonad indices declined by 50% over the 1500-fold range in adult population density. At 15 m deep locations, gonad mass was consistently low and did not vary significantly with density. Patterns of macroalgal abundance suggest urchins at high density and in deeper water were food limited. Because macroalgal cover co-varies inversely with sea urchin density, we designed field experiments to determine the interaction between sea urchin density and kelp canopy on fertilization success. On square arrays we manipulated the spacing of simulated urchins, but held their numbers constant (five sperm-filled syringes interspersed with four Nitex mesh egg containers permeable to sperm). These experiments, simulating the observed range of natural density, suggested that (1) fertilization rates decreased many times faster than individual gamete production increased over the same range in density, and (2) kelp increased fertilization success at high density when eggs were within 25 cm of a sperm source, but not when spaced 1 m apart. Additional laboratory fertilization experiments at ambient temperatures (3 to 5 °C) indicated that diluted sperm were viable for 8 h. In short, to the individual the reproductive benefits of aggregating appear to outweigh the costs; and while sperm may be limiting at low population density, eggs may remain viable long enough to be fertilized by sperm from more distant males.

Cosegregation of single genes associated with fertility restoration and transcript processing of sorghum mitochondrial orf107 and urf209.

Defective nuclear-cytoplasmic interactions leading to aberrant microgametogenesis in sorghum carrying the IS1112C male-sterile cytoplasm occur very late in pollen maturation. Amelioration of this condition, the restoration of pollen viability, involves a novel two-gene gametophytic system, wherein genes designated Rf3 and Rf4 are required for viability of individual gametes. Rf3 is tightly linked to, or represents, a single gene that regulates a transcript processing activity that cleaves transcripts of orf107, a chimeric mitochondrial open reading frame specific to IS1112C. The mitochondrial gene urf209 is also subject to nucleus-specific enhanced transcript processing, 5' to the gene, conferred by a single dominant gene designated Mmt1. Examinations of transcript patterns in F2 and two backcross populations indicated cosegregation of the augmented orf107 and urf209 processing activities in IS1112C. Several sorghum lines that do not restore fertility or confer orf107 transcript processing do exhibit urf209 transcript processing, indicating that the activities are distinguishable. We conclude that the nuclear gene(s) conferring enhanced orf107 and urf209 processing activities are tightly linked in IS1112C. Alternatively, the similarity in apparent regulatory action of the genes may indicate allelic differences wherein the IS1112C Rf3 allele may differ from alleles of maintainer lines by the capability to regulate both orf107 and urf209 processing activities.

The predetermination of embryonic sex using flow cytometrically separated X and Y spermatozoa

A review is given of the predetermination of sex in various domestic animals and in the human using sperm samples enriched for X- or Y-chromosome bearing spermatozoa obtained by flow cytometry and cell sorting. A comparison of other putative methods of sperm separation is made. In separating human X and Y spermatozoa, measurements of the DNA content in each individual gamete using the Hoechst fluorochrome 33342 remains the only validated method. The difference in DNA content between human X and Y spermatozoa is approximately 2.8%, and cell sorters have been adapted to take account of this and the asymmetrical nature of the sperm head. DNA analyses and PCR have been used to validate the method for animal spermatozoa. In the human, fluorescence in-situ hybridization (FISH) has confirmed sorting accuracy. Many correctly-diagnosed normal offspring have been born in various animal species and any potential mutagenic or cytotoxic effects are being closely monitored as are the cost and efficiency of the technology.

The iso1 gene of Chlamydomonas is involved in sex determination.

Sexual differentiation in the heterothallic alga Chlamydomonas reinhardtii is controlled by two mating-type loci, mt+ and mt-, which behave as a pair of alleles but contain different DNA sequences. A mutation in the mt minus-linked imp11 gene has been shown previously to convert a minus gamete into a pseudo-plus gamete that expresses all the plus gametic traits except the few encoded by the mt+ locus. Here we describe the iso1 mutation which is unlinked to the mt- locus but is expressed only in minus gametes (sex-limited expression). A population of minus gametes carrying the iso1 mutation behaves as a mixture of minus and pseudo-plus gametes: the gametes isoagglutinate but they do not fuse to form zygotes. Further analysis reveals that individual gametes express either plus or minus traits: a given cell displays one type of agglutinin (flagellar glycoprotein used for sexual adhesion) and one type of mating structure. The iso1 mutation identifies a gene unlinked to the mating-type locus that is involved in sex determination and the repression of plus-specific genes.

Sperm function and its manipulation for microassisted fertilization

Comprehension of the intricate complexities of sperm function is clearly crucial to the success of attempts to manipulate it for the purposes of assisted conception. This is particularly important when considering various procedures for microassisted fertilization since these bypass critical physiological events that are mandatory for normal fertilization, to varying degrees. Methylxanthine derivatives such as pentoxifylline are useful agents for the management of oligoasthenozoospermic patients. This is particularly so for procedures such as SUZI where adequate motility of spermatozoa injected into the perivitelline space is crucial for fusion with the vitelline membrane to achieve fertilization. The generation of minute concentrations of reactive oxygen species in vitro may prove to be a valuable technique in this respect, in the light of recent evidence for their involvement in capacitation and hyperactivation. Induction of the acrosome reaction by non-invasive, non-toxic agents should markedly improve success rates for microassisted fertilization. Acrosin appears to play a central role in this and, therefore, it would seem prudent to monitor levels of acrosin activity in samples of spermatozoa used in assisted conception procedures. With respect to microassisted fertilization, the potential to select recently acrosome-reacted spermatozoa coated by activated acrosin promises to be a major improvement. Current methods employed for determination of the fertilization potential of spermatozoa are clearly inadequate (Polansky and Lamb, 1988; Aitken, 1990). In fact, the prevailing evidence suggests that no single parameter of sperm function reflects this potential (Zaneveld and Jeyendran, 1988). Therefore, we have both a scientific and a moral responsibility to investigate these processes further. Subsequently, we should be in a position to identify individual gametes with the potential for fertilization and so utilize procedures that result in maximal fertilization rates with minimal risk of polyploidy or abnormality.

Desoxyribonucleic acid content of the egg of Sabellaria during maturation and fertilization.

THERE are at the present time no quantitative data on the deoxyribonucleic acid content of the individual gametes during maturation and fertilization. We have determined the deoxyribonucleic acid content of the egg of Sabellaria, from the first maturation division to the first segmentation division. As fertilization takes place before the first maturation division, both male and female components of the fertilized egg can be studied and compared. © 1951 Nature Publishing Group.