Cryopreservation of isolated micropores of spring rapeseed (Brassica napus L.) for in vitro embryo production

Microspore cryopreservation is a potentially powerful method for long-term storage of germplasm for in vitro embryo production in plant species. In this study, several factors influencing embryo production following the ultra-low temperature (–196 °C in liquid nitrogen) storage of isolated microspores of rapeseed (Brassica napus L.) were investigated. Microspores were prepared in cryogenic vials and subjected to various cooling treatments before immersion in liquid nitrogen for varying periods. Efficiency of microspore cryopreservation was reflected by in vitro embryo production from frozen microspores. Of all the cooling treatments, microspores treated with a cooling rate of 0.25% °C/min and a cooling terminal temperature of –35 °C before immersion in liquid nitrogen produced the highest embryo yields (18% and 40% of unfrozen controls in two genotypes, respectively). Fast thawing in a 35 °C water bath was necessary to recover a high number of embryos from microspore samples being frozen at a higher cooling rate, while thawing speed did not affect samples after freezing at a slower cooling rate. The storage density of cryopreserved microspores affected embryo production. Storage at the normal culture density (8×10⁴ microspores/ml) was less efficient for embryo production than at high densities (4×10⁶ microspores/ml and 1.6×10⁷ microspores/ml), although no significant difference was found between the high densities. Evaluation of plant lines derived from frozen microspores indicated no variation in isozyme pattern and no enhanced cold tolerance of these lines. Isolated microspores of B. napus could be stored for extended period for in vitro embryo production.     

Pink-eyed dilution alleles affect negative surface charges of mouse spermatozoa

Abnormal spermiogenesis in sterile pink-eyed dilution mutants results in spermatozoa with bizarre sperm heads. The spermatozoa of normal mice bind colloidal iron hydroxide (CIH) along the length of the tail, yet the spermatozoa of pink-eyed sterile mice show a great reduction in ability to bind CIH. This implies a loss of negative surface charges. The group(s) responsible for the charges are sensitive to methylation but resistant to neuraminidase treatment, even after deacetylation with alkaline treatment. The membrane components containing the negatively charged groups may be neuraminidase-resistant forms of gangliosides.     

Immunological approach to the characterization of the outer acrosomal membrane of boar spermatozoa

Antiserum to purified boar spermatozoan outer acrosomal membrane (OAM) was raised in rabbits and adsorbed with boar liver and serum glutaraldehyde cross-linked immunoadsorbents. The IgG fraction of the antiserum was purified by (NH₄)₂SO₄ precipitation followed by ion-exchange chromatography. Indirect immunofluorescence showed bright fluorescent staining of the acrosomal cap of boar spermatozoa and to a lesser extent of the acrosomes of bull and goat spermatozoa after incubation with anti-OAM-IgG. Immuno-electron microscopy further confirmed the specificity of the antibody for the OAM. Preincubation of the anti-OAM-IgG with isolated OAM, completely abolished its reactivity. When tested by ELISA, anti-OAM-IgG reacted with boar, bull, goat, and human spermatozoa; however, its binding activity to boar spermatozoa was significantly greater as compared to spermatozoa from the other species tested. In an effort to identify OAM antigens recognized by this antiserum, the isolated boar OAM was labeled either with ³H or with ¹²⁵I and solubilized by mild detergent treatment. The extracted components were immunoprecipitated with anti-OAM-IgG and protein A-bearing S. aureus and the thus isolated antigens were analysed on SDS-PAGE. The results suggest that anti-OAM-IgG recognized one high molecular ³H-labeled glycoprotein (270 kd), and four ¹²⁵I-labeled polypeptides of lower molecular weight of the boar OAM.     

Large-scale expansion of feeder-free mouse embryonic stem cells serially passaged in stirred suspension bioreactors at low inoculation densities directly from cryopreservation

Embryonic stem cells (ESCs) have almost unlimited proliferation capacity in vitro and can retain the ability to contribute to all cell lineages, making them an ideal platform material for cell-based therapies. ESCs are traditionally cultured in static flasks on a feeder layer of murine embryonic fibroblast cells. Although sufficient to generate cells for research purposes, this approach is impractical to achieve large quantities for clinical applications. In this study, we have developed protocols that address a variety of challenges that currently bottleneck clinical translation of ESCs expanded in stirred suspension bioreactors. We demonstrated that mouse ESCs (mESCs) cryopreserved in the absence of feeder cells could be thawed directly into stirred suspension bioreactors at extremely low inoculation densities (100 cells/ml). These cells sustained proliferative capacity through multiple passages and various reactor sizes and geometries, producing clinically relevant numbers (10⁹ cells) and maintaining pluripotency phenotypic and functional properties. Passages were completed in stirred suspension bioreactors of increasing scale, under defined batch conditions which greatly improved resource efficiency. Output mESCs were analyzed for pluripotency marker expression (SSEA-1, SOX-2, and Nanog) through flow cytometry, and spontaneous differentiation and teratoma analysis was used to demonstrate functional maintenance of pluripotency.     

Principles of viable preservation of parasitic Protozoa

Viable preservation is fundamentally different from serial passage as a way of maintaining Protozoa in the laboratory. In serial passage there is continuous selection of a reproducing population so that biological characteristics deviate from those representative of the original population towards those of a population adapted to the special conditions of the passage. Viable preservation arrests reproduction and so, this selection. Viably preserved material, therefore, deserves to be defined by a special term, ‘stabilate’, a stabilate being defined as a population of organisms viably preserved on a unique occasion. Materials maintained by serial passage are appropriately called ‘strains’.     

Some biochemical characteristics and preservation of epididymal camel spermatozoa (Camelus dromedarius)

Testicles were isolated from thirty five apparently healthy dromedary camels (Camelus dromedarius), aged between 5 to 18 years, in a local slaughterhouse during the rutting season. Epididymal fluid was collected from one epididymis for determination of twelve biochemical and antioxidant parameters using ELISA commercial kits. Spermatozoa were harvested from each region of the other epididymis (head, body and tail) and stored in SHOTOR^®, Green buffer^® + 20% egg yolk and INRA-96^® extenders at 5 and 30 °C. Results revealed that, in the epididymal fluid, concentrations of testosterone, glucose, albumin, total protein, cholesterol, fatty acids, iron, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were 5.19 ± 1.69 ng/mL, 3.10 ± 0.41 mmol/L, 6.26 ± 1.26 g/dL, 0.50 ± 0.07 mg/dL, 1.74 ± 0.09 mmol/L, 6.62 ± 0.81 nmol/ul, 926.20 ± 100.18 ug/dL, 51.17 ± 7.74 mIU/ml, and 143.16 ± 18.67 mIU/ml, respectively. The antioxidants activities of catalase, glutathione peroxidase (GPx) and superoxide dismutase (SOD) in the epididymal fluid were 121.55 ± 6.57 nmol/min/ml, 59.35 ± 10.98 nmol/min/ml and 0.18 ± 0.03 U/ml, respectively. Epididymal sperm motility and concentration were higher (P < 0.05) in the body and tail than the head. The viability indices of total and forward sperm motility, at 5 and 30 °C, obtained from the tail region were superior (P < 0.05) in both SHOTOR^® and INRA-96^® extenders than Green buffer extender. It may be concluded that INRA-96^® extender is the best for storing dromedary epididymal spermatozoa at 5 and 30 °C.     

Cryopreservation of the promastigote form of Leishmania tropica var major at different cooling rates

Callow L. L. and Farrant J. 1973. Cryopreservation of the promastigote form of Leishmania tropica var major at different cooling rates. International Journal for Parasitology, 3: 77–88. An investigation of the optimal conditions for the preservation of Leishmania tropica var major by freezing was undertaken because it was important to obtain a high yield when the thawed organisms were cultured. As a prerequisite for comparing different conditions, assay methods were devised. One method was based on the reduced growth of promastigotes in diphasic medium that was found to follow inoculation of relatively few organisms. The other employed serial ten-fold dilutions of suspensions of organisms, and the inoculation of medium at each dilution stage. Viability was related to the time taken for flagellates to be found in the medium. A 1·0 m solution of glycerol in the flagellate suspension inhibited growth when diphasic medium was inoculated. This effect was removed by separating the organisms from the glycerol before inoculation, or by diluting the suspension approximately one hundred-fold. A similar inhibition was not observed for dimethylsulphoxide (DMSO). Glycerol (1·0 m), DMSO (1·5 m), polyvinylpyrrolidone (10% w/v) and sucrose (0·3 m) were not obviously detrimental to the organisms. Normal growth in diphasic medium resulted when these additives were removed after being in contact with organisms for 5 h in an ice bath. In freezing experiments, flagellates survived freezing and thawing while they were still in contact with their nutrient medium, and also after they had been separated, washed and resuspended in isotonic saline with 10 mm of glucose. The survival rate was much greater when either 1·5 m DMSO or 1·0 m glycerol was added. These additives were compared at one rate only, $\text{0·3°C}\text{min}$, and DMSO gave better protection. With 1·5 m DMSO, maximal survival was obtained at a cooling rate of $\text{1·9°C}\text{min}$. Relatively high rates of cooling, that is, over $\text{400°C}\text{min}$ were detrimental to the organisms.     

Decreased expression of heat shock protein A4L in spermatozoa is positively related to poor human sperm quality

Heat shock protein A4L (HSPA4L), which is highly expressed in the testis, is correlated with male fertility. However, the relationship between HSPA4L expression and sperm quality remains unknown. In the present study, a systematic characterization of HSPA4L expression on spermatozoa was performed. HSPA4L is highly expressed in the human testis, characterized by abundant localization in testicular spermatocytes and round spermatids. Compared with the testis from young adults (aged 27–36 years old), downregulated expression of HSPA4L in the testis from elderly adults (aged 78–82 years old) was observed. Immunofluorescence quantification demonstrated the localization of HSPA4L in the middle piece of sperm. Compared with mature spermatozoa, a similar lower intensity and localization percentage of HSPA4L in immature and asthenozoospermic spermatozoa were observed, and the consistently decreased expression of HSPA4L in immature and asthenozoospermic spermatozoa was validated by western blot analysis. Functional analysis revealed a correlation between HSPA4L and sperm motility by Spearman correlation analysis and its involvement in sperm–oocyte penetration by the human sperm–hamster egg penetration test. The current study demonstrates that HSPA4L is a promising marker for the assessment of sperm quality and provides clues for exploring biomarkers for the molecular diagnosis and treatment of male infertility.     

Morphology of the male reproductive system of freshwater prawn Macrobrachium carcinus (Decapoda,Caridea): Functional and comparative aspects

Testes and vasa deferentia are parts of the male reproductive system of decapod crustaceans. Both organs show morphological differences among decapod species in terms of anatomical and histological patterns reflecting the diversity of this group. Describing these features may assist in systematics, phylogenetics, and studies of reproductive behavior, especially for species of commercial interest, such as Macrobrachium carcinus, a native American species that, unusually for this genus, has no precopulation courting behavior. This study aims to describe the reproductive morphology and spermatogenesis of the male freshwater prawn M. carcinus. The male reproductive system of this species consisted of lobed testes connected to the vasa deferentia. The testis of M. carcinus was divided into several lobules. Each lobule was formed by a cluster of germ cells surrounded by connective tissue and nurse cells. This microscopic anatomy and histology of the testicular histoarchitecture has been described for many species of Decapoda and may represent a derived design of the testes. Unlike that in other decapod species, spermatogenesis proceeds in short transitory phases that produce spermatozoa at high concentrations and frequencies, corroborating the uncommon male reproductive behavior of this species. In the spermatic pathway, the lobules develop and fuse before releasing spermatozoa from the testes; however, this process has not been observed in decapods, yet. The neutral compounds secreted by the vas deferens are important for sperm nutrition as females secrete a substance for spermatophore adhesion during reproduction. This study presents different features and dynamics of the spermatogenic process in the male reproductive system of M. carcinus that have not yet been presented in the literature for decapods.     

Heat stress,a serious threat to reproductive function in animals and humans

Global warming represents a major stressful environmental condition that compromises the reproductive efficiency of animals and humans via a rise of body temperature above its physiological homeothermic point (heat stress [HS]). The injuries caused by HS on reproductive function involves both male and female components, fertilization mechanisms as well as the early and late stages of embryo‐fetal development. This occurrence causes great economic damage in livestock, and, in wild animals creates selective pressure towards the advantages of better‐adapted genotypes to the detriment of others. Humans undergo several types of stress, including heat, and these represent putative causes of ongoing progressive decay in procreation; an increasing number of remedies in the form of antioxidant preparations are now being proposed to counteract the effects of stress. This review aims to describe the results of the most recent studies that aimed to highlight these effects and to draw information on the mechanisms acting as the basis of this problem from a comparative analysis.