Three-dimensional in vitro follicle growth: overview of culture models,biomaterials, design parameters and future directions

In vitro ovarian follicle culture is a new frontier in assisted reproductive technology with tremendous potential, especially for fertility preservation. Folliculogenesis within the ovary is a complex process requiring interaction between somatic cell components and the oocyte. Conventional two-dimensional culture on tissue culture substrata impedes spherical growth and preservation of the spatial arrangements between oocyte and surrounding granulosa cells. Granulosa cell attachment and migration can leave the oocyte naked and unable to complete the maturation process. Recognition of the importance of spatial arrangements between cells has spurred research in to three-dimensional culture system. Such systems may be vital when dealing with human primordial follicles that may require as long as three months in culture. In the present work we review pertinent aspects of in vitro follicle maturation, with an emphasis on tissue-engineering solutions for maintaining the follicular unit during the culture interval. We focus primarily on presenting the various 3-dimensional culture systems that have been applied for in vitro maturation of follicle:oocyte complexes. We also try to present an overview of outcomes with various biomaterials and animal models and also the limitations of the existing systems.     

Frequent and efficient use of the sister chromatid for DNA double-strand break repair during budding yeast meiosis

Recombination between homologous chromosomes of different parental origin (homologs) is necessary for their accurate segregation during meiosis. It has been suggested that meiotic inter-homolog recombination is promoted by a barrier to inter-sister-chromatid recombination, imposed by meiosis-specific components of the chromosome axis. Consistent with this, measures of Holliday junction-containing recombination intermediates (joint molecules [JMs]) show a strong bias towards inter-homolog and against inter-sister JMs. However, recombination between sister chromatids also has an important role in meiosis. The genomes of diploid organisms in natural populations are highly polymorphic for insertions and deletions, and meiotic double-strand breaks (DSBs) that form within such polymorphic regions must be repaired by inter-sister recombination. Efforts to study inter-sister recombination during meiosis, in particular to determine recombination frequencies and mechanisms, have been constrained by the inability to monitor the products of inter-sister recombination. We present here molecular-level studies of inter-sister recombination during budding yeast meiosis. We examined events initiated by DSBs in regions that lack corresponding sequences on the homolog, and show that these DSBs are efficiently repaired by inter-sister recombination. This occurs with the same timing as inter-homolog recombination, but with reduced (2- to 3-fold) yields of JMs. Loss of the meiotic-chromosome-axis-associated kinase Mek1 accelerates inter-sister DSB repair and markedly increases inter-sister JM frequencies. Furthermore, inter-sister JMs formed in mek1Δ mutants are preferentially lost, while inter-homolog JMs are maintained. These findings indicate that inter-sister recombination occurs frequently during budding yeast meiosis, with the possibility that up to one-third of all recombination events occur between sister chromatids. We suggest that a Mek1-dependent reduction in the rate of inter-sister repair, combined with the destabilization of inter-sister JMs, promotes inter-homolog recombination while retaining the capacity for inter-sister recombination when inter-homolog recombination is not possible.     

Fatty acids can substitute the HIV fusion peptide in lipid merging and fusion: an analogy between viral and palmitoylated eukaryotic fusion proteins

Various fusion proteins from eukaryotes and viruses share structural similarities such as a coiled coil motif. However, compared with eukaryotic proteins, a viral fusion protein contains a fusion peptide (FP), which is an N-terminal hydrophobic fragment that is primarily involved in directing fusion via anchoring the protein to the target cell membrane. In various eukaryotic fusion proteins the membrane targeting domain is cysteine-rich and must undergo palmitoylation prior to the fusion process. Here we examined whether fatty acids can replace the FP of human immunodeficiency virus type 1 (HIV-1), thereby discerning between the contributions of the sequence versus hydrophobicity of the FP in the lipid-merging process. For that purpose, we structurally and functionally characterized peptides derived from the N terminus of HIV fusion protein - gp41 in which the FP is lacking or replaced by fatty acids. We found that fatty acid conjugation dramatically enhanced the capability of the peptides to induce lipid mixing and aggregation of zwitterionic phospholipids composing the outer leaflet of eukaryotic cell membranes. The enhanced effect of the acylated peptides on membranes was further supported by real-time atomic force microscopy (AFM) showing nanoscale holes in zwitterionic membranes. Membrane-binding experiments revealed that fatty acid conjugation did not increase the affinity of the peptides to the membrane significantly. Furthermore, all free and acylated peptides exhibited similar α-helical structures in solution and in zwitterionic membranes. Interestingly, the fusogenic active conformation of N36 in negatively charged membranes composing the inner leaflet of eukaryotic cells is β-sheet. Apparently, N-terminal heptad repeat (NHR) can change its conformation as a response to a change in the charge of the membrane head group. Overall, the data suggest an analogy between the eukaryotic cysteine-rich domains and the viral fusion peptide, and mark the hydrophobic nature of FP as an important characteristic for its role in lipid merging.     

The Himalayas as a directional barrier to gene flow

High-resolution Y-chromosome haplogroup analyses coupled with Y-short tandem repeat (STR) haplotypes were used to (1) investigate the genetic affinities of three populations from Nepal--including Newar, Tamang, and people from cosmopolitan Kathmandu (referred to as "Kathmandu" subsequently)--as well as a collection from Tibet and (2) evaluate whether the Himalayan mountain range represents a geographic barrier for gene flow between the Tibetan plateau and the South Asian subcontinent. The results suggest that the Tibetans and Nepalese are in part descendants of Tibeto-Burman-speaking groups originating from Northeast Asia. All four populations are represented predominantly by haplogroup O3a5-M134-derived chromosomes, whose Y-STR-based age (+/-SE) was estimated at 8.1+/-2.9 thousand years ago (KYA), more recent than its Southeast Asian counterpart. The most pronounced difference between the two regions is reflected in the opposing high-frequency distributions of haplogroups D in Tibet and R in Nepal. With the exception of Tamang, both Newar and Kathmandu exhibit considerable similarities to the Indian Y-haplogroup distribution, particularly in their haplogroup R and H composition. These results indicate gene flow from the Indian subcontinent and, in the case of haplogroup R, from Eurasia as well, a conclusion that is also supported by the admixture analysis. In contrast, whereas haplogroup D is completely absent in Nepal, it accounts for 50.6% of the Tibetan Y-chromosome gene pool. Coalescent analyses suggest that the expansion of haplogroup D derivatives--namely, D1-M15 and D3-P47 in Tibet--involved two different demographic events (5.1+/-1.8 and 11.3+/-3.7 KYA, respectively) that are more recent than those of D2-M55 representatives common in Japan. Low frequencies, relative to Nepal, of haplogroup J and R lineages in Tibet are also consistent with restricted gene flow from the subcontinent. Yet the presence of haplogroup O3a5-M134 representatives in Nepal indicates that the Himalayas have been permeable to dispersals from the east. These genetic patterns suggest that this cordillera has been a biased bidirectional barrier.     

Identification of tissue-specific DNA-protein binding sites by means of two-dimensional electrophoretic mobility shift assay display

We developed a technique of differential electrophoretic mobility shift assay (EMSA) display allowing identification of tissue-specific protein-binding sites within long genomic sequences. Using this approach, we identified 10 cell type-specific protein-binding sites (protein target sites [PTSs]) within a 137-kb human chromosome 19 region. In general, tissue-specific binding of proteins from different nuclear extracts by individual PTSs did not follow the all-or-nothing principle. Most often, PTS-protein complexes were formed in all cases, but they were different for different nuclear extracts used.     

Electrophysical characteristics of Azospirillum brasilense Sp245 during interaction with antibodies to various cell surface epitopes

This work was undertaken to examine the electrooptical characteristics of cells of Azospirillum brasilense Sp245 during their interaction with antibodies developed to various cell surface epitopes. We used the dependences of the cell suspension optical density changes induced by electroorientation on the orienting field frequency (740, 1000, 1450, 2000, and 2800kHz). Cell interactions with homologous strain-specific antibodies to the A. brasilense Sp245 O antigen and with homologous antibodies to whole bacterial cells brought about considerable changes in the electrooptical properties of the bacterial suspension. When genus-specific antibodies to the flagellin of the Azospirillum sheathed flagellum and antibodies to the serologically distinct O antigen of A. brasilense Sp7 were included in the A. brasilense Sp245 suspension, the changes caused in the electrooptical signal were slight and had values close to those for the above changes. These findings agree well with the immunochemical characteristics of the Azospirillum O antigens and with the data on the topographical distribution of the Azospirillum major cell surface antigens. The obtained results can serve as a basis for the development of a rapid test for the intraspecies detection of microorganisms.     

Role of genetic and environmental influences on heart rate variability in middle-aged men

Our aim was to estimate causal relationships of genetic factors and different specific environmental factors in determination of the level of cardiac autonomic modulation, i.e., heart rate variability (HRV), in healthy male twins and male twins with chronic diseases. The subjects were 208 monozygotic (MZ, 104 healthy) and 296 dizygotic (DZ, 173 healthy) male twins. A structured interview was used to obtain data on lifetime exposures of occupational loading, regularly performed leisure-time sport activities, coffee consumption, smoking history, and chronic diseases from 12 yr of age through the present. A 5-min ECG at supine rest was recorded for the HRV analyses. In univariate statistical analyses based on genetic models with additive genetic, dominance genetic, and unique environmental effects, genetic effects accounted for 31-57% of HRV variance. In multivariate statistical analysis, body mass index, percent body fat, coffee consumption, smoking, medication, and chronic diseases were associated with different HRV variables, accounting for 1-11% of their variance. Occupational physical loading and leisure-time sport activities did not account for variation in any HRV variable. However, in the subgroup analysis of healthy and diseased twins, occupational loading explained 4% of the variability in heart periods. Otherwise, the interaction between health status and genetic effects was significant for only two HRV variables. In conclusion, genetic factors accounted for a major portion of the interindividual differences in HRV, with no remarkable effect of health status. No single behavioral determinant appeared to have a major influence on HRV. The effects of medication and diseases may mask the minimal effect of occupational loading on HRV.     

The ways of realization of high specificity and efficiency of enteropeptidase

Comparative substrate analysis of full-length bovine enteropeptidase and trypsin, bovine and human enteropeptidase light chains was performed using model N-terminal dodecapeptides corresponding to wild-type human trypsinogen and pancreatitis-associated mutant trypsinogens K23R and D22G. The substitution of Lys residue by Arg at P1 leads to 2-fold increase in the efficiency of enteropeptidase hydrolysis; the absence of the negatively charged residue at P2 reduces the efficiency of such hydrolysis by two orders of magnitude. The difference in efficiency of peptide chain hydrolysis after Lys/Arg residues by enteropeptidase compared to trypsin is equal to the difference in hydrolysis by serine proteases of different primary specificity of their specific substrates.