Slush nitrogen vitrification of human ovarian tissue does not alter gene expression and improves follicle health and progression in long-term in vitro culture.

OBJECTIVE: To study whether slush nitrogen (SN) vs. liquid nitrogen (LN) vitrification affects human ovarian tissue gene expression and preserves follicle health during extended in vitro culture. DESIGN: Randomized experimental study. SETTING: University research laboratory. PATIENT(S): Ovarian biopsies collected by laparoscopic surgery from patients with benign gynaecologic conditions. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Ovarian strips were vitrified with LN or SN, warmed, and analyzed before or after culture for 9 days (d9) in gas-permeable dishes. Expression of genes involved in stress and toxicity pathways was analyzed in fresh and warmed strips by polymerase chain reaction (PCR) array and quantitative real-time-PCR. Fresh and vitrified/warmed strips were analyzed for follicle quality, progression, and viability before or after culture. RESULT(S): The SN vitrification preserved follicle quality better than LN (% grade 1 follicles: fresh control, 54.2; LN, 29.3; SN, 48.8). Quantitative reverse transcription-PCR demonstrated a noticeable up-regulation of 13 genes in LN samples (range, 10-35) and a markedly lower up-regulation of only 5 genes (range, 3.6-7.8) in SN samples. Long-term in vitro culture evidenced worse follicle quality and viability in LN samples than in both fresh and SN samples (% grade 1 follicle: fresh d0, 51.5; fresh d9, 41; LN d9, 16.4; SN d9, 55) and a highly significant reduction of primordial follicles and a concomitant increase of primary and secondary follicles in all samples. Follicle growth to the secondary stage was significantly higher in vitrified tissue than in fresh tissue, being better in SN than in LN vitrified tissue. CONCLUSION(S): Follicle quality, gene expression, viability, and progression are better preserved after SN vitrification.

Is oxygen availability a limiting factor for in vitro folliculogenesis?

Transplantation of ovarian tissue for the preservation of fertility in oncological patients is becoming an accepted clinical practice. However, the risk of re-introducing tumour cells at transplantation has stirred an increased interest for complete in vitro folliculogenesis. This has not yet been achieved in humans possibly for the lack of knowledge on the environmental milieu that orchestrates folliculogenesis in vivo. The main aim of this study was to investigate the effect of oxygen availability on follicle health and growth during in vitro culture of ovarian tissue strips. To this end, a model was developed to predict the dissolved oxygen concentration in tissue under varying culture conditions. Ovarian cortical strips of bovine, adopted as an animal model, and human tissue were cultured in conventional (CD) and gas permeable (PD) dishes under different media column heights and gaseous oxygen tensions for 3, 6 and 9 days. Follicle quality, activation of primordial follicles to the primary stage, and progression to the secondary stage were analysed through histology. Follicle viability was assessed through a live-dead assay at the confocal scanning laser microscope. Findings showed a higher follicle quality and viability after culture of bovine ovarian strips in PD in adequate medium height and oxygen tensions. The best culture conditions found in the bovine were adopted for human ovarian strip culture and promoted a higher follicle quality, viability and progression. Overall, data demonstrated that modulation of oxygen availability in tissue plays a key role in maintaining follicles' health and their ability to survive and progress to the secondary stage during ovarian tissue in vitro culture. Such culture conditions could increase the yield of healthy secondary follicles for subsequent dissection and individual culture to obtain competent oocytes.

Supplementation of sperm media with zinc, D-aspartate and co-enzyme Q10 protects bull sperm against exogenous oxidative stress and improves their ability to support embryo development.

High levels of reactive oxygen species in the semen of infertile patients or spontaneously generated during in vitro sperm handling may impair sperm quality, fertilization and embryo developmental competence. We recently reported that zinc, d-aspartate and co-enzyme Q10, contained in the dietary supplement Genadis® (Merck Serono), have protective effects on human and bull sperm motility, lipid peroxidation and DNA fragmentation in vitro; furthermore, in bovine, treated spermatozoa had an improved ability to support embryo development. However, only a few studies have investigated the protective role of antioxidants during in vitro sperm handling in the presence of an exogenous oxidative stress. Herein, to simulate such conditions in an animal model, we induced exogenous oxidative stress on spermatozoa through the xanthine-xanthine oxidase system and investigated its effects on sperm function and subsequent embryo developmental competence in the presence of zinc, d-Asp and CoQ10 protection. The main results showed that exogenous oxidative stress decreased sperm motility, increased sperm DNA fragmentation, and reduced fertilization and blastocyst rates and quality. Pre-treatment with zinc, d-aspartate and co-enzyme Q10 before exogenous oxidative stress was able to prevent these effects. Supplementation of sperm culture media with zinc, d-aspartate and co-enzyme Q10 could protect sperm from oxidative stress damage during in vitro handling in assisted reproductive technologies.

Successful slush nitrogen vitrification of human ovarian tissue.

OBJECTIVE: To study whether slush nitrogen vitrification improves the preservation of human ovarian tissue. DESIGN: Control vs. treatment study. SETTING: University research laboratory. PATIENT(S): Ovarian biopsies collected from nine women (aged 14-35 years) during laparoscopic surgery for benign gynecologic conditions. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Ovarian cortical strips of 2 × 5 × 1 mm were vitrified with liquid or slush nitrogen. Fresh and vitrified cortical strips were analyzed for cryodamage and viability under light, confocal, and transmission electron microscopy. RESULT(S): Compared with liquid nitrogen, vitrification with slush nitrogen preserves [1] follicle quality (grade 1 follicles: fresh control, 50%; liquid nitrogen, 27%; slush nitrogen, 48%); [2] granulosa cell ultrastructure (intact cells: fresh control, 92%; liquid nitrogen, 45%; slush nitrogen, 73%), stromal cell ultrastructure (intact cells: fresh control, 59.8%; liquid nitrogen, 24%; slush nitrogen, 48.7%), and DNA integrity (TUNEL-positive cells: fresh control, 0.5%; liquid nitrogen, 2.3%; slush nitrogen, 0.4%); and [3] oocyte, granulosa, and stromal cell viability (oocyte: fresh control, 90%; liquid nitrogen, 63%; slush nitrogen, 87%; granulosa cells: fresh control, 93%; liquid nitrogen, 53%; slush nitrogen, 81%; stromal cells: fresh control, 63%; liquid nitrogen, 30%; slush nitrogen, 52%). CONCLUSION(S): The histology, ultrastructure, and viability of follicles and stromal cells are better preserved after vitrification with slush nitrogen compared with liquid nitrogen.

Adapting human videofluoroscopic swallow study methods to detect and characterize dysphagia in murine disease models.

This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research. Successful outcomes are dependent upon three critical components: test chambers that permit self-feeding while standing unrestrained in a confined space, recipes that mask the aversive taste/odor of commercially-available oral contrast agents, and a step-by-step test protocol that permits quantification of swallow physiology. Elimination of one or more of these components will have a detrimental impact on the study results. Moreover, the energy level capability of the fluoroscopy system will determine which swallow parameters can be investigated. Most research centers have high energy fluoroscopes designed for use with people and larger animals, which results in exceptionally poor image quality when testing mice and other small rodents. Despite this limitation, we have identified seven VFSS parameters that are consistently quantifiable in mice when using a high energy fluoroscope in combination with the new murine VFSS protocol. We recently obtained a low energy fluoroscopy system with exceptionally high imaging resolution and magnification capabilities that was designed for use with mice and other small rodents. Preliminary work using this new system, in combination with the new murine VFSS protocol, has identified 13 swallow parameters that are consistently quantifiable in mice, which is nearly double the number obtained using conventional (i.e., high energy) fluoroscopes. Identification of additional swallow parameters is expected as we optimize the capabilities of this new system. Results thus far demonstrate the utility of using a low energy fluoroscopy system to detect and quantify subtle changes in swallow physiology that may otherwise be overlooked when using high energy fluoroscopes to investigate murine disease models.

Energy independent uptake and release of polystyrene nanoparticles in primary mammalian cell cultures.

Nanoparticle (NPs) delivery systems in vivo promises to overcome many obstacles associated with the administration of drugs, vaccines, plasmid DNA and RNA materials, making the study of their cellular uptake a central issue in nanomedicine. The uptake of NPs may be influenced by the cell culture stage and the NPs physical-chemical properties. So far, controversial data on NPs uptake have been derived owing to the heterogeneity of NPs and the general use of immortalized cancer cell lines that often behave differently from each other and from primary mammalian cell cultures. Main aims of the present study were to investigate the uptake, endocytosis pathways, intracellular fate and release of well standardized model particles, i.e. fluorescent 44 nm polystyrene NPs (PS-NPs), on two primary mammalian cell cultures, i.e. bovine oviductal epithelial cells (BOEC) and human colon fibroblasts (HCF) by confocal microscopy and spectrofluorimetric analysis. Different drugs and conditions that inhibit specific internalization routes were used to understand the mechanisms that mediate PS-NP uptake. Our data showed that PS-NPs are rapidly internalized by both cell types 1) with similar saturation kinetics; 2) through ATP-independent processes, and 3) quickly released in the culture medium. Our results suggest that PS-NPs are able to rapidly cross the cell membrane through passive translocation during both uptake and release, and emphasize the need to carefully design NPs for drug delivery, to ensure their selective uptake and to optimize their retainment in the targeted cells.

Protective effects of in vitro treatment with zinc, d-aspartate and coenzyme q10 on human sperm motility, lipid peroxidation and DNA fragmentation.

BACKGROUND: Spermatozoa are extremely vulnerable to oxidative stress caused by the unbalance between concentrations of reactive oxygen species and antioxidant scavenging systems present inside the male reproductive tract. In spite of a large number of clinical studies that claimed the beneficial effects of antioxidant oral administration on sperm physiology and fertility, only a few studies were addressed to evaluate their effects on spermatozoa in vitro. Main aims of the present study were to assess the influence of zinc, D-aspartate and coenzyme Q10, included in the dietary supplement Genadis (Merck Serono), on human sperm motility, DNA fragmentation and lipid peroxidation. METHODS: Semen samples, obtained from forty-four patients (23-30 years of age) were enrolled in this study, twenty-four were normospermic and twenty patients were oligospermic. Semen samples were analysed for sperm progressive motility and kinetics through computer assisted analysis, DNA fragmentation and lipid peroxidation. RESULTS: Main results showed that in both normo and oligospermic samples, total and progressive sperm motility is maintained by in vitro treatment with zinc, D-aspartate and coenzyme Q10, whereas a significant decrease of these parameters occurs in parallel samples incubated in medium alone. Zinc, D-aspartate and coenzyme Q10 also prevented the decrease of sperm kinetics but such an effect was highly significant only in oligospermic samples. Moreover, they also protected spermatozoa by the increase of DNA fragmentation and lipid peroxidation. CONCLUSIONS: Zinc, D-aspartate and coenzyme Q10 exert a direct protective effect on human spermatozoa preventing the decrease of motility and the increase of DNA fragmentation and lipid peroxidation during in vitro culture.

Red blood with blue-blood ancestry: intriguing structure of a snail hemoglobin.

The phylogenetic enigma of snail hemoglobin, its isolated occurrence in a single gastropod family, the Planorbidae, and the lack of sequence data, stimulated the present study. We present here the complete cDNA and predicted amino acid sequence of two hemoglobin polypeptides from the planorbid Biomphalaria glabrata (intermediate host snail for the human parasite Schistosoma mansoni). Both isoforms contain 13 different, cysteine-free globin domains, plus a small N-terminal nonglobin "plug" domain with three cysteines for subunit dimerization (total M(r) approximately 238 kDa). We also identified the native hemoglobin molecule and present here a preliminary 3D reconstruction from electron microscopical images (3 nm resolution); it suggests a 3 x 2-mer quaternary structure (M(r) approximately 1.43 MDa). Moreover, we identified a previously undescribed rosette-like hemolymph protein that has been mistaken for hemoglobin. We also detected expression of an incomplete hemocyanin as trace component. The combined data show that B. glabrata hemoglobin evolved from pulmonate myoglobin, possibly to replace a less-efficient hemocyanin, and reveals a surprisingly simple evolutionary mechanism to create a high molecular mass respiratory protein from 78 similar globin domains.