Sperm mitochondrial DNA transmission to both male and female offspring in the blue mussel Mytilus galloprovincialis

In Mytilus mussels, paternal mitochondrial DNA (M type) from sperm is known to be transmitted to offspring. This phenomenon is called doubly uniparental inheritance (DUI). Under DUI, it has been reported that female mussels generally have only maternal mtDNA (F type). In this study, we examined the mode of mtDNA transmission in Mytilus galloprovincialis using M and F type-specific primer sets. The ratio of M and F types were measured in each sample by SNaPshot. The M type was detected in the adductor muscle and female gonad of all females. In unfertilized eggs spawned by 84.6% of females (22/26), M type was also detected. The F type was more abundant than the M type in all females. Although the ratio of M type in females was very low, all females contained the M type. From these results, we propose a new possibility about DUI inheritance. The presence of M type in unfertilized eggs indicates that the M type of eggs may also contribute to M type inheritance.     

The exceptional mitochondrial DNA system of the mussel family Mytilidae

Species of the families Mytilidae (sea mussels) and Unionidae (fresh water mussels) contain two types of mitochondrial DNA (mtDNA), the F that behaves as the standard animal mtDNA and the M that is transmitted through the sperm and establishes itself only in the male gonad. The two molecules have, therefore, separate transmission routes, one through the female and the other through the male lineage. The system has been named doubly uniparental inheritance (DUI). Another important feature of sea mussels is that the sex ratio among offspring of a pair mating is determined by the female parent only. The mechanism of DUI remains unknown. One hypothesis that is consistent with all observations is that the standard maternal inheritance was modified in mussels via the evolution of a suppressor gene that is expressed during oogenesis and has two alleles, the inactive and the active allele. In the presence of the active allele in the mother's genotype the egg is supplied with a substance that interferes and the normal mechanism of elimination of sperm mitochondria. This will explain why half of mussels have the father's mtDNA and half do not, but would not explain why presence/absence of paternal mtDNA is linked with the male and female gender, respectively. To provide an explanation for this linkage, one would have to assume that there is a causal relationship between retention of paternal mtDNA and sex determination.     

Quantitation of the male and female types of mitochondrial DNA in a blue mussel, Mytilus galloprovincialis, using real-time polymerase chain reaction assay

The system termed doubly uniparental inheritance (DUI) of mitochondrial transmission to progeny has been reported in Mytilus. Under DUI, it has been thought that males have both paternally (M type) and maternally (F type) transmitted mitochondrial DNA (mtDNA), and females have only F type. However, the presence of M type in females has been reported. To clarify the ratio of M type to F type mtDNA in female and male tissues to further our understanding of mitochondrial transmission, we developed a procedure to measure the copy numbers of the two types of mtDNA in Mytilus galloprovincialis using a real-time polymerase chain reaction assay. The following results were obtained by this method. In females, the copy numbers of M type mtDNA detected in adductor muscle, gonad and eggs were approximately 10 000-fold lower than those of F type. In males, F type dominated in adductor muscle, as in the female tissue. However, copy numbers of M type mtDNA were approximately 1000-fold higher than those of F type in gonad and 100 000-fold higher than those of F type in sperm. We examined the quantity relationship between the two types of mtDNA and the transmission mechanism of mtDNA in M. galloprovincialis.     

Sex-specific effects of hybridization on reproductive fitness in Mytilus

Blue mussels of the genus Mytilus form extensive hybrid zones in the North Atlantic and elsewhere where the distributions of different species overlap. Mytilus species transmit both maternal and paternal mtDNA through egg and sperm, respectively, a process known as doubly uniparental inheritance (DUI), and some females produce offspring with extremely biased sex ratios. These two traits have been shown to be linked and maternally controlled, with sex determination involving nuclear–cytoplasmic interactions. Hybridization has been shown to disrupt DUI mitochondrial inheritance and sex ratio bias; however, the effect of hybridization on reproductive fitness has not previously been examined. We investigated this effect in M. edulis × M. trossulus crosses through histological examination of mature F1 progeny, and spawning of F1 hybrids to monitor survival of their progeny through to the D stage of larval development. For progeny produced from mothers with a strong bias toward female offspring (often 100%) in pure-bred crosses, there was a clear breakdown in female dominance of progeny and significantly more hermaphrodites in the hybrid crosses produced from sperm with the M-tr1 mitotype. We also found significant sex-specific differences among hybrid progeny, with females producing normal eggs while males and hermaphrodites evidenced impaired gonadal development with significantly greater numbers of Sertoli cells, phagocytic hemocytes, and degenerating germ cells, all associated with gonad resorption. Males from crosses where DUI was disrupted and where male progeny were homoplasmic for the female mtDNA were the most severely compromised. Allelic incongruity between maternal and paternal mitotypes in hybrid crosses was associated with significant disruption of male gonadal development.     

The Effects of Natural Hybridization on the Regulation of Doubly Uniparental Mtdna Inheritance in Blue Mussels (Mytilus Spp.)

Blue mussels in the Mytilus edulis species complex have a doubly uniparental mode of mtDNA inheritance with separate maternal and paternal mtDNA lineages. Female mussels inherit their mtDNA solely from their mother, while males inherit mtDNA from both parents. In the male gonad the paternal mtDNA is preferentially replicated so that only paternal mtDNA is transmitted from fathers to sons. Hybridization is common among differentiated blue mussel taxa; whenever it involves M. trossulus, doubly uniparental mtDNA inheritance is disrupted. We have found high frequencies of males without and females with paternal mtDNA among hybrid mussels produced by interspecific matings between M. galloprovincialis and M. trossulus. In contrast, hybridization between M. galloprovincialis and M. edulis does not affect doubly uniparental inheritance, indicating a difference in the divergence of the mechanisms regulating mtDNA inheritance among the three blue mussel taxa. Our data indicate a high frequency of disrupted mtDNA transmission in F(1) hybrids and suggest that two separate mechanisms, one regulating the transmission of paternal mtDNA to males and another inhibiting the establishment of paternal mtDNA in females, act to regulate doubly uniparental inheritance. We propose a model for the regulation of doubly uniparental inheritance that is consistent with these observations.     

Maternal inheritance of mitochondrial DNA (mtDNA) in the Pacific oyster (Crassostrea gigas): a preliminary study using mtDNA sequence analysis with evidence of random distribution of MitoTracker-stained sperm mitochondria in fertilized eggs

In many bivalve species, paternal and maternal mitochondrial DNA (mtDNA) from sperm and eggs is transmitted to the offspring. This phenomenon is known as doubly uniparental inheritance (DUI). In these species, sperm mtDNA (M type) is inherited by the male gonad of the offspring. Egg mtDNA (F type) is inherited by both male and female somatic cells and female gonadal cells. In Mytilidae, sperm mitochondria are distributed in the cytoplasm of differentiating male germ cells because they are transmitted to the male gonad. In the present study, we investigated maternal inheritance of mtDNA in the Pacific oyster, Crassostrea gigas. Sequence analysis of two mitochondrial non-coding regions revealed an identical sequence pattern in the gametes and adductor muscle samples taken from six males and five females. To observe whether sperm mitochondria were specifically located in the cytoplasm of differentiating germ cells, their distribution was recorded in C. gigas fertilized eggs by vital staining with MitoTracker Green. Although the 1D blastomere was identified in the cytoplasm of differentiating germ cells, sperm mitochondria were located at the 1D blastomere in only 32% of eggs during the 8-cell stage. Thus, in C. gigas, sperm mitochondria do not specifically locate in the germ cell region at the 1D blastomere. We suggest that the distribution of sperm mitochondria is not associated with germ cell formation in C. gigas. Furthermore, as evidenced by the mtDNA sequences of two non-coding regions, we conclude that mitochondrial DNA is maternally inherited in this species.     

No evidence for presence of maternal mitochondrial DNA in the sperm of Mytilus galloprovincialis males

Species of the mussel family Mytilidae have a special mitochondrial DNA (mtDNA) transmission system, known as doubly uniparental inheritance (DUI), which consists of a maternally inherited (F) and a paternally inherited (M) mitochondrial genome. Females are normally homoplasmic for the F genome and males are heteroplasmic mosaics, with their somatic tissues dominated by the maternal and their gonads dominated by the paternal genome. Several studies have indicated that the maternal genome may often be present in the male germ line. Here we report the results from the examination of mtDNA in pure sperm from more than 30 males of Mytilus galloprovincialis. In all cases, except one, we detected only the M genome. In the sperm of one male, we detected a paternal genome with an F-like primary sequence that was different from the sequence of the maternal genome in the animal's somatic tissues. We conclude that the male germ line is protected against invasion by the maternal genome. This is important because fidelity of gamete-specific transmission of the two mitochondrial genomes is a basic requirement for the stability of DUI.     

Disruption of doubly uniparental inheritance of mitochondrial DNA in hybrid mussels (Mytilus edulis x M. galloprovincialis)

Blue mussels of the genus Mytilus have an unusual mode of mitochondrial DNA inheritance termed doubly uniparental inheritance (DUI). Females are homoplasmic for the F mitotype which is inherited maternally, whereas males are heteroplasmic for this and the paternally inherited M mitotype. In areas where species distributions overlap a varying degree of hybridization occurs; yet genetic differences between allopatric populations are maintained. Observations from natural populations and previous laboratory experiments suggest that DUI may be disrupted by hybridization, giving rise to heteroplasmic females and homoplasmic males. We carried out controlled laboratory crosses between Mytilus edulis and M. galloprovincialis to produce pure species and hybrid larvae of known parentage. DNA markers were used to follow the fate of the F and M mitotypes through larval development. Disruption of the mechanism which determines whether the M mitotype is retained or eliminated occurred in an estimated 38% of M. edulis x M. galloprovincialis hybrid larvae, a level double that previously observed in adult mussels from a natural M. edulis x M. galloprovincialis hybrid population. Furthermore, reciprocal hybrid crosses exhibited contrasting types of DUI disruption. The results indicate that disruption of DUI in hybrid mussels may be associated with increased mortality and hence could be a factor in the maintenance of genetic integrity for each species.     

Phylogenetic evidence for role-reversals of gender-associated mitochondrial DNA in Mytilus (Bivalvia: Mytilidae)

Distinct gender-associated mitochondrial DNA (mtDNA) lineages (i.e., lineages which are transmitted either through males or through females) have been demonstrated in two families of bivalves, the Mytilidae (marine mussels) and the Unionidae (freshwater mussels), which have been separated for more than 400 Myr. The mode of transmission of these M (for male-transmitted) and F (for female-transmitted) molecules has been referred to as doubly uniparental inheritance (DUI), in contrast to standard maternal inheritance (SMI), which is the norm in animals. A previous study suggested that at least three origins of DUI are required to explain the phylogenetic pattern of M and F lineages in freshwater and marine mussels. Here we present phylogenetic evidence based on partial sequences of the cytochrome c oxidase subunit I gene and the 16S RNA gene that indicates the DUI is a dynamic phenomenon. Specifically, we demonstrate that F lineages in three species of Mytilus mussels, M. edulis, M. trossulus, and M. californianus, have spawned separate lineages which are now associated only with males. This process is referred to as "masculinization" of F mtDNA. By extension, we propose that DUI may be a primitive bivalve character and that periodic masculinization events combined with extinction of previously existing M types effectively reset the time of divergence between conspecific gender-associated mtDNA lineages.      

Maternal inheritance of mouse mtDNA in interspecific hybrids: segregation of the leaked paternal mtDNA followed by the prevention of subsequent paternal leakage.

The transmission profiles of sperm mtDNA introduced into fertilized eggs were examined in detail in F1 hybrids of mouse interspecific crosses by addressing three aspects. The first is whether the leaked paternal mtDNA in fertilized eggs produced by interspecific crosses was distributed stably to all tissues after the eggs'' development to adults. The second is whether the leaked paternal mtDNA was transmitted to the subsequent generations. The third is whether paternal mtDNA continuously leaks in subsequent backcrosses. For identification of the leaked paternal mtDNA, we prepared total DNA samples directly from tissues or embryos and used PCR techniques that can detect a few molecules of paternal mtDNA even in the presence of 10(8)-fold excess of maternal mtDNA. The results showed that the leaked paternal mtDNA was not distributed to all tissues in the F1 hybrids or transmitted to the following generations through the female germ line. Moreover, the paternal mtDNA leakage was limited to the first generation of an interspecific cross and did not occur in progeny from subsequent backcrosses. These observations suggest that species-specific exclusion of sperm mtDNA in mammalian fertilized eggs is extremely stringent, ensuring strictly maternal inheritance of mtDNA.     

Heteroplasmy suggests paternal co-transmission of multiple genomes and pervasive reversion of maternally into paternally transmitted genomes of mussel (Mytilus) mitochondrial DNA

Marine mussels of the genus Mytilus have two types of mitochondrial DNA with separate paternal and maternal inheritance. Females are homoplasmic for an F genome that is transmitted to all offspring, whereas males are heteroplasmic for this F genome and for a highly diverged (> 20%) M genome that is transmitted only to sons. Here we provide phylogenetic evidence based on lrRNA sequence data that most of the paternal genomes in European M. trossulus have an introgressive female M. edulis origin and are nearly indistinguishable in sequence from F types of M. trossulus. This observation is best explained by the hypothesis that introgressed F type molecules have recently invaded the paternal route and have assumed the role of M molecules, then resetting to zero the time of sequence divergence between M and F lineages. European M. trossulus shows a high prevalence of males heteroplasmic for three different mitochondrial DNA types all having the same two paternal types and the same maternal type, consistent with paternal co-transmission of multiple genomes. Co-transmission of the same genomes must apparently operate uninterruptedly for several generations in spite of the very different evolutionary origin of the specific molecules that are transmitted paternally and maternally in European M. trossulus.     

Male-Dependent Doubly Uniparental Inheritance of Mitochondrial DNA and Female-Dependent Sex-Ratio in the Mussel Mytilus Galloprovincialis

We have investigated sex ratio and mitochondrial DNA inheritance in pair-matings involving five female and five male individuals of the Mediterranean mussel Mytilus galloprovincialis. The percentage of male progeny varied widely among families and was found to be a characteristic of the female parent and independent of the male to which it was mated. Thus sex-ratio in Mytilus appears to be independent of the nuclear genotype of the sperm. With a few exceptions, doubly uniparental inheritance (DUI) of mtDNA was observed in all families fathered by four of the five males: female and male progeny contained the mother's mtDNA (the F genome), but males contained also the father's paternal mtDNA (the M genome). Two hermaphrodite individuals found among the progeny of these crosses contained the F mitochondrial genome in the female gonad and both the F and M genomes in the male gonad. All four families fathered by the fifth male showed the standard maternal inheritance (SMI) of animal mtDNA: both female and male progeny contained only the maternal mtDNA. These observations illustrate the intimate linkage between sex and mtDNA inheritance in species with DUI and suggest different major roles for each gender. We propose a model according to which development of a male gonad requires the presence in the early germ cells of an agent associated with sperm-derived mitochondria, these mitochondria are endowed with a paternally encoded replicative advantage through which they overcome their original minority in the fertilized egg and this advantage (and, therefore, the chance of an early entrance into the germ line) is countered by a maternally encoded egg factor.     

Fate of maternal mtDNA following 60Co inactivation of maternal nuclear DNA in unfertilized salmonid eggs.

The effects of gamma irradiation on nuclear DNA and mitochondrial DNA (mtDNA) were examined by exposing unfertilized salmonid eggs to a 60Co source. Brown trout (Salmo trutta) eggs exposed to 60Co were fertilized with sperm from brook trout (Salvelinus fontinalis), and brook trout eggs exposed to 60Co were fertilized with sperm from splake males (S. namaycush x S. fontinalis). In both types of matings only paternal allozymes were found in embryos, confirming the inactivation of the nuclear genome in the eggs. Analysis of mtDNA in these same embryos showed exclusively maternal mtDNA. The absence of paternal mtDNA among any of the embryos supports the predominance of maternal inheritance of mtDNA in vertebrates and suggests that mtDNAs are more resistant to cobalt inactivation than nuclear DNAs based on structure or numerical superiority to maternal nuclear DNA. Inactivation of maternal nuclear DNA, fertilization, and an induced return to the diploid state provide a means for producing an inbred organism having the nuclear genome of the paternal parent (androgenetic) and the mitochondrial genome of the female.     

Doubly uniparental inheritance of mitochondrial DNA in freshwater mussels: History and status of the European species

Doubly uniparental inheritance (DUI) is a mode of inheriting mitochondrial DNA that is distinct from strictly maternal inheritance. It has been described in nine and three families of marine and freshwater mussels, respectively, including the European margaritiferids and unionids. Among the 16 freshwater species of Unionida inhabiting Europe, DUI has been described in 9 species of dioecious mussels and was absent from a single hermaphroditic species and from secondary hermaphroditic specimens. The DUI freshwater mussels include two vastly genetically different mitochondrial genomes: maternal (F genome) and paternal (M genome), which coexist within the same specimen but in different tissues. The F genome is present in all female tissues and somatic male tissues. It is inherited in the typical, maternal, manner. Conversely, the M genome is located primarily in the male gonads and generative cells, and is inherited paternally. Dioecious Unionidae display unique characteristics that have been interrelated for over 200 million years: a high fidelity of the transmission of the F and M genomes in DUI and two paths of spermatogenesis–the typical path that produces sperm cells containing mitochondria with the F genome and the atypical path that produces sperm cells with the M genome. The mitogenomes of freshwater mussels display unique features that are not present in any other animal, that is, an additional, gender-specific gene and an elongated cox2 gene occurring exclusively in the M genome. These features mean that the mitochondria, in addition to their basic function of producing energy, also may take part in determining sex in these dioecious organisms.     

Patterns of polymorphism and gene flow of gender-associated mitochondrial DNA lineages in European mussel populations

Mussels of the genus Mytilus have two types of mitochondrial DNA (mtDNA). The M type is transmitted paternally and the F type is transmitted maternally. RFLP analysis is used to assess phylogenetic relationships and nucleotide diversity and divergence for both mtDNA genomes in European populations of M. edulis and Atlantic and Mediterranean forms of M. galloprovincialis. Ten restriction endonucleases were used to assay variation in regions of the ND2 and COIII genes for a total of 77 individuals. F and M genomes show a concordant phylogenetic split into two major divergent clades, one specific to Mediterranean M. galloprovincialis and the other containing haplotypes from the three taxa. For both genomes, the geographical distribution of mtDNA variation suggests: (i) extensive levels of mtDNA introgression; (ii) asymmetric mtDNA gene flow from Atlantic to Mediterranean populations; and (iii) recurrent historical hybridization events. Significantly higher mtDNA diversity and divergence are observed for the M than F genome in all three Mytilus taxa, although the evolutionary forces responsible for these differences cannot be resolved. The extensive mtDNA gene flow between European Mytilus taxa conflicts with the restricted mtDNA introgression observed in American mussels , implying geographical variation in the nature of nuclear/mtDNA interactions regulating biparental inheritance.     

Doubly uniparental inheritance of mitochondrial DNA: Might it be simpler than we thought?

More than 100 species of bivalve mollusks are currently known to carry two highly diverged mitochondrial DNA (mtDNA) molecules, one of which is transmitted through the egg and the other through the sperm generation after generation, faithfully and uninterruptedly. This mtDNA system, which has become known as doubly uniparental inheritance (DUI), is most likely unique in eukaryotes and constitutes a striking deviation from the strictly maternal inheritance (SMI) of mtDNA that is the rule in the animal kingdom. Here, I present a model of how the paternal mtDNA may escape the mitochondrial destruction that occurs prior to sperm formation and enter the male germ line in the newly formed embryo. In essence, the model treats the sperm-transmitted mtDNA as a molecule that takes a ride with the sperm. The model can be easily tested and, if passed the tests, may open the way for the understanding of DUI at the molecular level and throw light on the mechanisms and evolution of mtDNA transmission in general. In addition, the model shifts attention from nuclear control of paternal mtDNA inheritance, whether systematic (as DUI) or leaky (as the cases reported in a wide variety of animal species), to the mtDNA itself as the protagonist of its own transmission. This possibility has been, so far, ignored in studies of paternal mtDNA transmission in other species including humans.     

Comparative analysis of gender-associated complete mitochondrial genomes in marine mussels (Mytilus spp.)

Marine mussels of the genus Mytilus have an unusual mode of mitochondrial DNA (mtDNA) transmission termed doubly uniparental inheritance (DUI). Female mussels are homoplasmic for the F mitotype, which is inherited maternally, while males are usually heteroplasmic, carrying a mixture of the maternal F mitotype and the paternally inherited M genome. Two classes of M genomes have been observed: "standard" M genomes and "recently masculinized" M genomes. The latter are more similar to F genomes at the sequence level but are transmitted paternally like standard M genomes. In this study we report the complete sequences of two standard male M. edulis and one recently masculinized male M. trossulus mitochondrial genome. A comparative analysis, including the previously sequenced M. edulis F and M. galloprovincialis F and M mtDNAs, reveals that these genomes are identical in gene order, but highly divergent in nucleotide and amino acid sequence. The large amount (>20%) of nucleotide substitutions that fall in coding regions implies that there are several amino acid replacements between the F and M genomes, which likely have an impact on the structural and functional properties of the mitochondrial proteome. Correlation of the divergence rate of different protein-coding genes indicates that mtDNA-encoded proteins of the M genome are still under selective constraints, although less highly than genes of the F genome. The mosaic F/M control region of the masculinized F genome provides evidence for lineage-specific sequences that may be responsible for the different mode of transmission genetics. This analysis shows the value of comparative genomics to better understand the mechanisms of maintenance and segregation of mtDNA sequence variants in mytilid mussels.     

Evolutionary relationships among the male and female mitochondrial DNA lineages in the Mytilus edulis species complex

A novel form of mitochondrial DNA (mtDNA) inheritance has previously been documented for the blue mussel (Mytilus edulis). Female mussels inherit their mtDNA solely from their mother while males inherit mtDNA from both their mother and their father. In males, the paternal mtDNA is preferentially amplified so that the male gonad is highly enriched for the paternal mtDNA that is then transmitted from fathers to sons. We demonstrate that this mode of mtDNA inheritance also operates in the closely related species M. galloprovincialis and M. trossulus. The evolutionary relationship between the male and female mtDNA lineages is estimated by phylogenetic analysis of 455 nucleotides from the large subunit ribosomal RNA gene. We have found that the male and female lineages are highly divergent; the divergence of these lineages began prior to the speciation of the three species of blue mussels. Further, the separation between the male and female lineages is estimated to have occurred between 5.3 and 5.7 MYA.