Mayu Obata

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.

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.

Different transcriptional ratios of male and female transmitted mitochondrial DNA and tissue‐specific expression patterns in the blue mussel, Mytilus galloprovincialis

In some bivalve species, paternal mitochondrial DNA (mtDNA) from sperm is transmitted to the offspring. This is called “doubly uniparental inheritance” (DUI). Under DUI, male offspring receive both paternal (M type) and maternal (F type) mtDNA. Females predominantly receive F type. Expression levels of M and F type mtDNA and mitochondrial RNA localization have not been studied extensively. In this study, we quantified M and F type mtDNA and their expression levels in male and female somatic tissues and gonads with real‐time polymerase chain reaction (PCR) in the blue mussel, Mytilus galloprovincialis. M and F type expression patterns were studied with in situ hybridization, using probes specific to M and F type mtDNA in the cytochrome b region. We found that (i) F type mtDNA was expressed in somatic tissues and female gonads, while M type was not expressed in these tissues; (ii) M type expression in male gonads was limited, but strong expression was observed during early spermatogenesis; and (iii) F type expression ratios were significantly lower in female gonads than in somatic tissues and lower than both M and F type expression ratios in male gonads. We propose (i) different systems for M and F type tissue‐specific transcriptional regulation; and (ii) different functions for F and M type mtDNA, with F type being functional in somatic tissues and female gonads and M type functioning only in spermatogenetic cells.

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.

Pearl Microstructure and Expression of Shell Matrix Protein Genes MSI31 and MSI60 in the Pearl Sac Epithelium of Pinctada fucata by In Situ Hybridization

Expression patterns of the shell matrix protein genes MSI31 and MSI60 in the pearl sac epithelium were examined by in situ hybridization 38 days after implantation, and related to pearl quality. A pearl sac that produced a nacreous pearl showed very weak expression of MSI31 and strong expression of MSI60. A pearl sac, which yielded a prismatic pearl, strongly expressed MSI31 and very weakly expressed MSI60. In a complex pearl, whose surface consisted of a mosaic of both nacreous and prismatic layers, the expression pattern of MSI31 and MSI60 similarly corresponded to the underlying surface structures of the pearl. A nacreous pearl whose pearl sac showed strong MSI31 expression had an entirely nacreous surface composed of a laminar structure with unusual tablet growth at the corresponding site. MSI31 and MSI60 are the major components of the shell matrix proteins of the nacreous and prismatic layers. Clearly, high expression of MSI31 does not always result in prismatic secretion. These observations cannot be explained solely on the basis of the expression patterns of MSI31 and MSI60. We propose that, in addition to the MSI genes that form the prismatic and nacreous layers, upstream from these genes there are regulatory master genes that determine whether a nacreous layer (aragonite) or a prismatic layer (calcite) is formed.

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 proliferation and migration of immature germ cells in the mussel, Mytilus galloprovincialis : observation of the expression pattern in the M. galloprovincialis vasa -like gene ( Myvlg ) by in situ hybridization

In bivalve, the distribution of primordial germ cells can be traced from early embryogenesis to the veliger larva by the expression of the vasa ortholog. However, the distribution of germ cells from metamorphosis to maturation in bivalves has not been examined extensively. In this study, we used in situ hybridization to observe expression of the Mytilus galloprovincialis vasa-like gene (Myvlg). The distribution of germ cells was clarified in immature mussels. We observed germ cells in adult mussels during the non-reproductive and reproductive seasons. Myvlg was specifically expressed in germ cells. Gametogenesis occurs in acini surrounded by connective tissue. Myvlg expression was detected in spermatogonia, spermatocytes, oogonia, and oocytes. In the non-reproductive season, gametes were not observed in the acini, but Myvlg was expressed in germinal stem cells along the acini. The expression intensity in the non-reproductive season, however, was much weaker than that in the reproductive season. Myvlg-positive cells proliferated during the non-reproductive season. In immature mussels, a pair of germ cell clumps was distributed laterally in the connective tissue between the nephric tubules and posterior byssal retractor muscle. Germ cells were also observed along pericardium. When immature mussels grew, a pair of germ cell clumps migrated anteriorly in the connective tissue along the outer epithelium at the dorsal region of the mantle base between the mantle and gill. The number of germ cells increased significantly as the mussels grew. This is the first report to observe the proliferation and migration of germ cells in immature mussels.

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

Abstract 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.

A Hypothesis of Ploidy Elevation by Formation of a Female Pronucleus in the Androgenetic Clam Corbicula fluminea in the Tone River Estuary, Japan

Abstract We propose a hypothesis of ploidy elevation in the androgenetic clam Corbicula fluminea, based on an abnormal process of fertilization in clams collected at the Tone River, Ibaraki Prefecture, Japan. Most eggs showed androgenesis, that is, extrusion of all maternal chromosomes as two polar bodies during the first meiotic division. Most eggs did not form a female pronucleus, but only a male pronucleus. However, some eggs proceeded to the second meiosis and formed both a female and a male pronucleus. The formation of the female pronucleus suggests the hypothesis that ploidy elevation in androgenetic clams may have occurred by aberrant meiosis due to an altered orientation of the meiotic spindle.

Mitochondrial DNA Transmitted from Sperm in the Blue Mussel Mytilus galloprovincialis Showing Doubly Uniparental Inheritance of Mitochondria, Quantified by Real-Time PCR

Doubly uniparental inheritance (DUI) of mitochondrial DNA transmission to progeny has been reported in the mussel, Mytilus. In DUI, males have both paternally (M type) and maternally (F type) transmitted mitochondrial DNA (mtDNA), but females have only the F type. To estimate how much M type mtDNA enters the egg with sperm in the DUI system, ratios of M type to F type mtDNA were measured before and after fertilization. M type mtDNA content in eggs increased markedly after fertilization. Similar patterns in M type content changes after fertilization were observed in crosses using the same males. To compare mtDNA quantities, we subsequently measured the ratios of mtDNA to the 28S ribosomal RNA gene (an endogenous control sequence) in sperm or unfertilized eggs using a real-time polymerase chain reaction (PCR) assay. F type content in unfertilized eggs was greater than the M type in sperm by about 1000-fold on average. M type content in spermatozoa was greater than in unfertilized egg, but their distribution overlapped. These results may explain the post-fertilization changes in zygotic M type content. We previously demonstrated that paternal and maternal M type mtDNAs are transmitted to offspring, and hypothesized that the paternal M type contributed to M type transmission to the next generation more than the maternal type did. These quantitative data on M and F type mtDNA in sperm and eggs provide further support for that hypothesis.