Fertility preservation: improved neovascularization and follicle viability in cryopreserved bovine ovarian cortex transplants by remaining medulla tissue.

OBJECTIVE: To investigate the advantages of cryopreserved medulla-containing ovarian cortex grafts with those of commonly used sole cortex grafts for fertility preservation by analyzing tissue quality, neovascularization processes, and the number of vital follicles. DESIGN: Experimental setting of cryopreserved bovine ovarian cortex tissue grafts with or without medulla tissue. SETTING: Laboratory animal research at Ulm University, Ulm, Germany. ANIMALS: Bovine ovaries and fertilized chicken eggs. INTERVENTION(S): Experimental setting of bovine ovarian tissue samples grafted on the chicken chorioallantoic membrane (CAM) after cryopreservation and thawing to examine histologic tissue integrity, apoptosis and proliferation immunohistochemically, blood vessel counts and determine the presence of neutral red-stained vital follicles. MAIN OUTCOME MEASURE(S): We used hematoxylin and eosin staining to visualize tissue structures, immunohistochemistry with anti-caspase 3 to detect apoptosis, anti-Ki67 to examine proliferation, blood vessel count on the chicken CAM to investigate neovascularization processes, and neutral red staining to evaluate vital follicles. RESULT(S): We demonstrated that in all analyzed tissue samples, after cryopreservation, thawing, and grafting on the chicken CAM, there was excellent tissue integrity and quality, as shown by extremely rare apoptosis processes analyzed using immunohistochemical caspase 3 staining (sole cortex, 0.54%; thin medulla-containing cortex, 0.43%; thick medulla-containing cortex, 0.13%; and sole medulla, 2.82%). Moreover, we detected increased neovascularization in the vicinity of medulla and medulla-containing grafts (small blood vessels: cortex 8.7, thin medulla-containing cortex 9.9, thick medulla-containing cortex 9.7, and medulla 9.8; very small blood vessels: cortex 7.0, thin medulla-containing cortex 13.0, thick medulla-containing cortex 12.0, and medulla 15.0), with higher Ki67-detected proliferation (cortex, 17.58%; thin medulla-containing cortex, 20.28%; thick medulla-containing cortex, 20.56%; and medulla, 29.9%). Additionally, we identified an increased number of vital follicles in medulla-containing cortex grafts compared with the number of vital follicles in sole cortex tissue (cortex, 256.1; thin medulla-containing cortex, 338.2; thick medulla-containing cortex, 346.6; and medulla, 8.1). CONCLUSION(S): In this experimental setting, bovine medulla-containing cortex tissue had excellent tissue structure and quality after cryopreservation and thawing and increased neovascularization and an augmented vital follicle count after grafting than the commonly used sole cortex tissue. Therefore, we suggest reconsidering the current cryopreservation and grafting processes in humans for fertility preservation by favoring retain medulla tissue at the ovarian cortex.

Prokaryotic multicellularity: a nanopore array for bacterial cell communication.

The transition from unicellular to multicellular life, which occurred several times during evolution, requires tight interaction and communication of neighboring cells. The multicellular cyanobacterium Nostoc punctiforme ATCC 29133 forms filaments of hundreds of interacting cells exchanging metabolites and signal molecules and is able to differentiate specialized cells in response to environmental stimuli. Mutation of cell wall amidase AmiC2 leads to a severe phenotype with formation of aberrant septa in the distorted filaments, which completely lack cell communication and potential for cell differentiation. Here we demonstrate the function of the amidase AmiC2 in formation of cell-joining structures. The AmiC2 protein localizes to the young septum between cells and shows bona fide amidase activity in vivo and in vitro. Vancomycin staining identified the overall septum morphology in living cells. By electron microscopy of isolated peptidoglycan sacculi, the submicroscopic structure of the cell junctions could be visualized, revealing a novel function for a cell wall amidase: AmiC2 drills holes into the cross-walls, forming an array of ~155 nanopores with a diameter of ~20 nm each. These nanopores seem to constitute a framework for cell-joining proteins, penetrating the cell wall. The entire array of junctional nanopores appears as a novel bacterial organelle, establishing multicellularity in a filamentous prokaryote.

Cell wall amidase AmiC1 is required for cellular communication and heterocyst development in the cyanobacterium Anabaena PCC 7120 but not for filament integrity.

Filamentous cyanobacteria of the order Nostocales display typical properties of multicellular organisms. In response to nitrogen starvation, some vegetative cells differentiate into heterocysts, where fixation of N(2) takes place. Heterocysts provide a micro-oxic compartment to protect nitrogenase from the oxygen produced by the vegetative cells. Differentiation involves fundamental remodeling of the gram-negative cell wall by deposition of a thick envelope and by formation of a neck-like structure at the contact site to the vegetative cells. Cell wall-hydrolyzing enzymes, like cell wall amidases, are involved in peptidoglycan maturation and turnover in unicellular bacteria. Recently, we showed that mutation of the amidase homologue amiC2 gene in Nostoc punctiforme ATCC 29133 distorts filament morphology and function. Here, we present the functional characterization of two amiC paralogues from Anabaena sp. strain PCC 7120. The amiC1 (alr0092) mutant was not able to differentiate heterocysts or to grow diazotrophically, whereas the amiC2 (alr0093) mutant did not show an altered phenotype under standard growth conditions. In agreement, fluorescence recovery after photobleaching (FRAP) studies showed a lack of cell-cell communication only in the AmiC1 mutant. Green fluorescent protein (GFP)-tagged AmiC1 was able to complement the mutant phenotype to wild-type properties. The protein localized in the septal regions of newly dividing cells and at the neck region of differentiating heterocysts. Upon nitrogen step-down, no mature heterocysts were developed in spite of ongoing heterocyst-specific gene expression. These results show the dependence of heterocyst development on amidase function and highlight a pivotal but so far underestimated cellular process, the remodeling of peptidoglycan, for the biology of filamentous cyanobacteria.

The morphogene AmiC2 is pivotal for multicellular development in the cyanobacterium Nostoc punctiforme.

Filamentous cyanobacteria of the order Nostocales are primordial multicellular organisms, a property widely considered unique to eukaryotes. Their filaments are composed of hundreds of mutually dependent vegetative cells and regularly spaced N(2)-fixing heterocysts, exchanging metabolites and signalling molecules. Furthermore, they may differentiate specialized spore-like cells and motile filaments. However, the structural basis for cellular communication within the filament remained elusive. Here we present that mutation of a single gene, encoding cell wall amidase AmiC2, completely changes the morphology and abrogates cell differentiation and intercellular communication. Ultrastructural analysis revealed for the first time a contiguous peptidoglycan sacculus with individual cells connected by a single-layered septal cross-wall. The mutant forms irregular clusters of twisted cells connected by aberrant septa. Rapid intercellular molecule exchange takes place in wild-type filaments, but is completely abolished in the mutant, and this blockage obstructs any cell differentiation, indicating a fundamental importance of intercellular communication for cell differentiation in Nostoc. AmiC2-GFP localizes in the cell wall with a focus in the cross walls of dividing cells, implying that AmiC2 processes the newly synthesized septum into a functional cell-cell communication structure during cell division. AmiC2 thus can be considered as a novel morphogene required for cell-cell communication, cellular development and multicellularity.