Mitochondrial DNA polymorphisms and sperm motility in Mytilus edulis (Bivalvia: Mytilidae)

The system of mitochondrial DNA (mtDNA) inheritance in Mytilus and other bivalves, termed doubly uniparental inheritance (DUI), is novel among animals. Males pass on their male transmitted (M-type) mtDNA from fathers to their sons whereas females pass on their female transmitted (F-type) mtDNA from mothers to both sons and daughters. Thus, Mytilus males contain two distinct types of mtDNA. Interestingly, sperm contains only the paternal mtDNA. Phylogenetic analysis has shown that some female types have been able to switch their route of inheritance. These "recently masculinized" mitochondrial genomes behave as a typical M-type in that they are transmitted from generation to generation through sperm. Because the "recently masculinized" and "standard" male mitotypes in M. edulis exhibit approximately 8.7% amino acid sequence divergence, we hypothesized that these differences could affect mitochondrial, and hence sperm, functions. Furthermore, since recently masculinized mitotypes have been shown to replace standard male types periodically over evolutionary timescales, we tested the hypothesis that sperm swimming speeds would be greater for males with recently masculinized M-type genomes. Sperm activity was videotaped, digitized and tracked. A linear mixed effects model found no significant difference in linear velocities or curvilinear speeds between the mitotypes suggesting that swimming speeds are similar for both in the period shortly after spawning.     

Biparental Inheritance Through Uniparental Transmission: The Doubly Uniparental Inheritance (DUI) of Mitochondrial DNA

Many bivalvian mollusks have a sperm-transmitted mitochondrial genome (M), along with the standard egg-transmitted one (F). The phenomenon, known as doubly uniparental inheritance (DUI) of mtDNA, is the only known case in which biparental inheritance of a cytoplasmic genome is the rule rather than the exception. In the mussel Mytilus sperm mitochondria disperse randomly among blastomeres in female embryos, but form an aggregate and stay in the same blastomere in male embryos. In adults, somatic tissues of both sexes are dominated by the F genome. Sperm contains only the M genome and eggs the F (and perhaps traces of M). A female produces mostly daughters, mostly sons, or both sexes in about equal numbers, irrespective of its mate. Thus maleness and M mtDNA fate are tightly linked and under maternal control. Hybridization and triploidization affect the former but not the latter, which suggests that the two are not causally linked. Gene content and arrangement are the same in conspecific F and M genomes, but primary sequence has diverged from 20 % to 40 %, depending on species. The two genomes differ at the control region (CR). Synonymous substitutions accumulate faster in the M than the F genome and non-synonymous even faster. Expression studies indicate that the M genome is active only at spermatogenesis. These observations suggest that the M genome is under a more relaxed selective constraint than the F. Some mytilid species carry, in low frequencies, sperm-transmitted mtDNAs whose primary sequence is of the F type and the CR is an F/M mosaic (“masculinized” genomes). In venerids sperm mitochondria behavior, M genome fate and sex determination are as in mytilids. In unionids the M genome also evolves faster than the F and F/M sequence divergence reaches 50 %. The identification of F-specific and M-specific open reading frames in non-coding regions of unionids and mytilids, in conjunction with the CR’s mosaic structure of masculinized genomes, suggest that the mitochondrial genomes of species with DUI carry sequences that affect their transmission route. A model that incorporates these findings is presented in this review.     

Segregation of sperm mitochondria in two- and four-cell embryos of the blue mussel Mytilus edulis: Implications for the mechanism of doubly uniparental inheritance of mitochondrial DNA.

Species of the family Mytilidae have 2 mitochondrial genomes, one that is transmitted through the egg and one that is transmitted through the sperm. In the Mytilus edulis species complex (M. edulis, M. galloprovincialis, and M. trossulus) there is also a strong mother-dependent sex-ratio bias in favor of one or the other sex among progeny from pair matings. In a previous study, we have shown that sperm mitochondria enter the egg and that their behavior during cell division is different depending on whether the egg originated from a female- or male-biased mother. Specifically, in eggs from females that produce mostly or exclusively daughters, sperm mitochondria disperse randomly among cells after egg division. In eggs from females that produce predominantly sons, sperm mitochondria tend to stay together in the same cell. Here, we extend these observations and show that in 2- and 4-cell embryos from male-biased mothers most sperm mitochondria are located near or at the cleavage furrow of the major cell, in contrast to embryos from female-biased mothers where there is no preferential association of sperm mitochondria with the cleavage furrow. This observation provides evidence for an early developmental mechanism through which sperm mitochondria are preferentially channeled into the primordial cells of male embryos, thus making the paternal mitochondrial genome the dominant mtDNA component of the male germ line.     

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.     

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.     

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.     

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.     

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.     

Mitochondrial DNA copy number is maintained during spermatogenesis and in the development of male larvae to sustain the doubly uniparental inheritance of mitochondrial DNA system in the blue mussel Mytilus galloprovincialis

Doubly uniparental inheritance (DUI) of mitochondrial (mt) DNA has been reported in the blue mussel Mytilus galloprovincialis. In DUI, males inherit both paternal (M type) and maternal (F type) mtDNA. Here we investigated changes in M type mtDNA copy numbers and mitochondrial mass in testicular cells by real‐time polymerase chain reaction and flow cytometry. The ratios of M type mtDNA copy numbers to nuclear DNA content were not different between haploid (1n), diploid (2n) and tetraploid (4n) spermatogenic cells. The mitochondrial mass decreased gradually during spermatogenesis. These results suggest that mtDNA and mitochondrial mass are maintained during spermatogenesis. We then traced M type mtDNA in larvae after fertilization. M type mtDNA was maintained up to 24 h after fertilization in the male‐biased crosses, but decreased significantly in female‐biased crosses (predicted by Mito Tracker staining pattern). These results are strikingly different from those reported for mammals and fish, where it is well known that the mitochondria and mtDNA are reduced during spermatogenesis and that sperm mitochondria and mtDNA are eliminated soon after fertilization. Thus, the M type mtDNA copy number is maintained during spermatogenesis and in the development of male larvae to sustain the DUI system in the blue mussel.     

Inheritance of two M type mitochondrial DNA from sperm and unfertilized eggs to offspring in Mytilus galloprovincialis

In Mytilus mussels, paternal mitochondrial DNA (mtDNA) from sperm is known to be transmitted to offspring. This phenomenon is called doubly uniparental inheritance (DUI). Under DUI, sperm mtDNA (M type) is inherited only by males. Female mussels receive maternal mtDNA (F type). However, in our previous study, we showed female and unfertilized eggs have both F and M types. We hypothesized that the two M types both from sperm and unfertilized eggs were transmitted to offspring. To test the hypothesis, we examined the number of M type haplotypes in mature M. galloprovincialis. The M type in larvae was compared with those of the parents. Cross experiments were carried out to test the inheritance of M type. In six of 20 mature mussels, two M types were detected by sequence analysis and polymerase chain reaction-restriction fragment length polymorphism. In cross experiments of larval samples from five of 12 crosses, double peak wave was observed by single nucleotide polymorphisms analysis. In these larval samples, the higher peak wave was identical to the parental M type. Larvae received much more paternal M type than the maternal ones. We demonstrated that two M types from sperm and unfertilized eggs were transmitted to offspring in M. galloprovincialis.     

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.     

Sex-linked mitochondrial behavior during early embryo development in Ruditapes philippinarum (Bivalvia Veneridae) a species with the Doubly Uniparental Inheritance (DUI) of mitochondria

In most metazoans mitochondria are inherited maternally. However, in some bivalve molluscs, two mitochondrial lineages are present: one transmitted through females (F-type), the other through males (M-type). This unique system is called Doubly Uniparental Inheritance (DUI) of mitochondria. In DUI species, M-type mitochondria have to invade the germ line of male embryos during development, otherwise sperm would transmit F-type mtDNA and DUI would fail. The mechanisms by which sperm mitochondria enter the germ line are still unknown. To address this question, we traced the movement of spermatozoon mitochondria (M-type) in embryos of the DUI species Ruditapes philippinarum by fertilizing eggs with sperm stained with the mitochondrial-specific vital dye MitoTracker Green. As in Mytilus DUI species, in R. philippinarum the distribution of sperm mitochondria follows two different patterns: an aggregated one in which these organelles locate near the first cleavage furrow, and a dispersed one in which sperm mitochondria are scattered. The presence of the two mitochondrial patterns in these taxa, together with their absence in species with Strictly Maternal Inheritance (SMI), confirms that their occurrence is related to DUI. Moreover, a Real-Time qPCR analysis showed that neither M-type nor F-type mitochondria undergo replication boosts in the earliest embryo development. This is the first study on sex-linked mtDNA copy number carried out by qPCR analysis on embryos of a DUI species and the first time the segregation patterns of sperm mitochondria are described in a DUI system other than Mytilus.     

Tissue-specific expression of male-transmitted mitochondrial DNA and its implications for rates of molecular evolution in Mytilus mussels (Bivalvia: Mytilidae).

Mytilus and other bivalves exhibit an unusual system of mitochondrial DNA (mtDNA) transmission termed doubly uniparental inheritance (DUI). Specifically, males transmit the mtDNA they have received from their fathers to their sons. Females transmit their mother's mtDNA to both sons and daughters. Males are normally heteroplasmic and females are normally homoplasmic, but not exclusively. This system is associated with an unusual pattern of molecular evolution. Male-transmitted mtDNA (M type) evolves faster than female-transmitted (F type) mtDNA. Relatively relaxed selection on the M type has been proposed as an explanation for this phenomenon. To further evaluate the selective forces acting upon the M-type genome, we used RT-PCR to determine where it is expressed. M-type mtDNA expression was detected in all gonad samples and in 50% of somatic tissues of males, and in a single female tissue. F-type mtDNA expression was detected in all female tissues, all male somatic tissues, and all but one male gonad sample. We argue that the expression of M-type mtDNA in male somatic and male gonad tissues has implications for the strength of selection acting upon it.     

Strict sex-specific mtDNA segregation in the germ line of the DUI species Venerupis philippinarum (Bivalvia: Veneridae)

Doubly Uniparental Inheritance (DUI) is one of the most striking exceptions to the common rule of standard maternal inheritance of metazoan mitochondria. In DUI, two mitochondrial genomes are present, showing different transmission routes, one through eggs (F-type) and the other through sperm (M-type). In this paper, we report results from a multiplex real-time quantitative polymerase chain reaction analysis on the Manila clam Venerupis philippinarum (formerly Tapes philippinarum). We quantified M- and F-types in somatic tissues, gonads, and gametes. Nuclear and external reference sequences were used, and the whole experimental process was designed to avoid any possible cross-contamination. In most male somatic tissues, the M-type is largely predominant: This suggests that the processes separating sex-linked mitochondrial DNAs (mtDNAs) in somatic tissues are less precise than in other DUI species. In the germ line, we evidenced a strict sex-specific mtDNA segregation because both sperm and eggs do carry exclusively M- and F-types, respectively, an observation that is in contrast with a previous analysis on Mytilus galloprovincialis. More precisely, whereas two mtDNAs are present in the whole gonad, only the sex-specific one is detected in gametes. Because of this, we propose that the mtDNA transmission is achieved through a three-checkpoint process in V. philippinarum. The cytological mechanisms of male mitochondria segregation in males and degradation in females during the embryo development (here named Checkpoint #1 and Checkpoint #2) are already well known for DUI species; a Checkpoint #3 would act when primordial germ cells (PGCs) are first formed and would work in both males and females. We believe that Checkpoint #3 is a mere variation of the "mitochondrial bottleneck" in species with standard maternal inheritance, established when their PGCs separate during embryo cleavage.     

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.     

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.     

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.     

Specific location of sperm mitochondria in mussel Mytilus galloprovincialis zygotes stained by MitoTracker

In Mytilidae, mitochondrial DNA (mtDNA) in the offspring is inherited from male and female parents. Sperm mitochondria are only incorporated into the testes. This phenomenon is called doubly uniparental inheritance (DUI). Sperm mitochondria should locate in the primordial germ cell during development to maintain DUI. However, the mechanism of sperm mitochondria localization is still unknown. To reveal the mechanism, we followed the location of sperm mitochondria in Mytilus galloprovincialis zygotes fertilized with sperm stained by MitoTracker. Just after fertilization, sperm mitochondria, which were found to enter eggs from various sites, remained at sperm entry point. Five sperm mitochondria located at the male pronucleus. After pronuclear expansion, sperm mitochondria migrated to the center of the egg together with the male pronucleus. At anaphase of cleavage-I, the distribution pattern of sperm mitochondria was divided into two patterns. In pattern A, sperm mitochondria located in the equatorial region of the eggs. In pattern B, sperm mitochondria migrated and divided into two groups with chromosomes. From observations of colchicine-treated eggs, we suggest that sperm mitochondria migration from fertilization to anaphase of cleavage-I depends on the microtubules. The difference between pattern A and pattern B may be caused by whether sperm mitochondria migrated or not by the microtubules at cleavage-I.