VASA-induced cytoplasmic localization of CYTB-positive mitochondrial substance occurs by destructive and nondestructive mitochondrial effusion, respectively, in early and late spermatogenic cells of the Manila clam

To analyze the release of mitochondrial material, a process that is believed to be (i) induced by the VASA protein derived from germplasm granules, and (ii) which appears to play an important role during meiotic differentiation, the localization of the CYTB protein was studied in the process of spermatogenesis of the bivalve mollusk Ruditapes philippinarum (Manila clam). It was found that in early spermatogenic cells, such as spermatogonia and spermatocytes, the CYTB protein shows dispersion in the cytoplasm following the total disaggregation of VASA-invaded mitochondria, what is called here as \u201cdestructive mitochondrial effusion (DME).\u201d It was found that the mitochondria of the maturing sperm cells also uptake VASA. It is accompanied by extramitochondrial transmembrane localization of CYTB assuming mitochondrial content release without mitochondrion demolishing. This phenomenon is called here as \u201cnondestructive mitochondrial effusion (NDME).\u201d Thus, in the spermatogenesis of the Manila clam, two patterns of mitochondrial release, DME and NDME, were found, which function, respectively, in early spermatogenic cells and in maturing spermatozoa. Despite the morphological difference, it is assumed that both DME and NDME have a similar functional nature. In both cases, the intramitochondrial localization of VASA coincides with the extramitochondrial localization of the mitochondrial matrix

Multiparametric optical bioimaging reveals the fate of epoxy crosslinked biomeshes in the mouse subcutaneous implantation model

Biomeshes based on decellularized bovine pericardium (DBP) are widely used in reconstructive surgery due to their wide availability and the attractive biomechanical properties. However, their efficacy in clinical applications is often affected by the uncontrolled immunogenicity and proteolytic degradation. To address this issue, we present here in vivo multiparametric imaging analysis of epoxy crosslinked DBPs to reveal their fate after implantation. We first analyzed the structure of the crosslinked DBP using scanning electron microscopy and evaluated proteolytic stability and cytotoxicity. Next, using combination of fluorescence and hypoxia imaging, X-ray computed microtomography and histology techniques we studied the fate of DBPs after subcutaneous implantation in animals. Our approach revealed high resistance to biodegradation, gradual remodeling of a surrounding tissue forming the connective tissue capsule and calcification of crosslinked DBPs. These changes were concomitant to the development of hypoxia in the samples within 3 weeks after implantation and subsequent induction of angiogenesis and vascularization. Collectively, presented approach provides new insights on the transplantation of the epoxy crosslinked biomeshes, the risks associated with its applications in soft-tissue reconstruction and can be transferred to studies of other types of implants

Subcellular Distribution of Mitochondrial Ribosomal RNA in the Mouse Oocyte and Zygote

Mitochondrial ribosomal RNAs (mtrRNAs) have been reported to translocate extra-mitochondrially and localize to the germ cell determinant of oocytes and zygotes in some metazoa except mammals. To address whether the mtrRNAs also localize in the mammals, expression and distribution of mitochondrion-encoded RNAs in the mouse oocytes and zygotes was examined by whole-mount in situ hybridization (ISH). Both 12S and 16S rRNAs were predominantly distributed in the animal hemisphere of the mature oocyte. This distribution pattern was rearranged toward the second polar body in zygotes after fertilization. The amount of mtrRNAs decreased around first cleavage, remained low during second cleavage and increased after third cleavage. Staining intensity of the 12S rRNA was weaker than that of the 16S rRNA throughout the examined stages. Similar distribution dynamics of the 16S rRNA was observed in strontium-activated haploid parthenotes, suggesting the distribution rearrangement does not require a component from sperm. The distribution of 16S rRNAs did not coincide with that of mitochondrion-specific heat shock protein 70, suggesting that the mtrRNA is translocated from mitochondria. The ISH-scanning electron microscopy confirms the extra-mitochondrial mtrRNA in the mouse oocyte. Chloramphenicol (CP) treatment of late pronuclear stage zygotes perturbed first cleavage as judged by the greater than normal disparity in size of blastomeres of 2-cell conceptuses. Two-third of the CP-treated zygotes arrested at either 2-cell or 3-cell stage even after the CP was washed out. These findings indicate that the extra-mitochondrial mtrRNAs are localized in the mouse oocyte and implicated in correct cytoplasmic segregation into blastomeres through cleavages of the zygote

Doubly Uniparental Inheritance of Mitochondria As a Model System for Studying Germ Line Formation

BACKGROUND: Doubly Uniparental Inheritance (DUI) of mitochondria occurs when both mothers and fathers are capable of transmitting mitochondria to their offspring, in contrast to the typical Strictly Maternal Inheritance (SMI). DUI was found in some bivalve molluscs, in which two mitochondrial genomes are inherited, one through eggs, the other through sperm. During male embryo development, spermatozoon mitochondria aggregate in proximity of the first cleavage furrow and end up in the primordial germ cells, while they are dispersed in female embryos. METHODOLOGY/PRINCIPAL FINDINGS: We used MitoTracker, microtubule staining and transmission electron microscopy to examine the mechanisms of this unusual distribution of sperm mitochondria in the DUI species Ruditapes philippinarum. Our results suggest that in male embryos the midbody deriving from the mitotic spindle of the first division concurs in positioning the aggregate of sperm mitochondria. Furthermore, an immunocytochemical analysis showed that the germ line determinant Vasa segregates close to the first cleavage furrow. CONCLUSIONS/SIGNIFICANCE: In DUI male embryos, spermatozoon mitochondria aggregate in a stable area on the animal-vegetal axis: in organisms with spiral segmentation this zone is not involved in cleavage, so the aggregation is maintained. Moreover, sperm mitochondria reach the same embryonic area in which also germ plasm is transferred. In 2-blastomere embryos, the segregation of sperm mitochondria in the same region with Vasa suggests their contribution in male germ line formation. In DUI male embryos, M-type mitochondria must be recognized by egg factors to be actively transferred in the germ line, where they become dominant replacing the Balbiani body mitochondria. The typical features of germ line assembly point to a common biological mechanism shared by DUI and SMI organisms. Although the molecular dynamics of the segregation of sperm mitochondria in DUI species are unknown, they could be a variation of the mechanism regulating the mitochondrial bottleneck in all metazoans

Nuage constituents arising from mitochondria: Is it possible?

An ultrastructural study of nuage-mitochondria complexes in spermatogonia of the sea urchin, Anthocidaris crassispina, was carried out. Release of mitochondrial contents into the cytoplasm was observed. The mitochondrial derivatives persisted as cristae-containing globules of friable material that subsequently contacted and integrated with nuage. The present ultrastructural findings agree with the results of other researchers who proposed that germ plasm substance probably produced by the nucleus is supplemented by the mitochondrial genome.link_to_subscribed_fulltex

Observation of Cells Not Showing Hypersensitive Reaction in the Central Part of Tobacco Mosaic Virus (TMV)-Induced Local Lesions Developing in Detached Leaves of Datura stramonium L.

It is established that the central area of TMV-induced local lesions developed in detached Datura stramonium leaves, along with the completely collapsed cells (types I and II), contains cells (type III) conserving to a certain degree integrity of their structural components. A characteristic of the type III cells was the accumulation of considerable amount of virus and formation of TMV-specific granular and tubular inclusions. The study of lesion development showed that a proportion of the collapsed cells and cells of type III did not essentially change in the period up from 3 to 5 days after infection of the leaves. These data suggest that the disease development in cells of type III does not lead to a hypersensitive response and is very similar to that in the systemically infected cells