Long-term preservation of eri and ailanthus silkworms using frozen gonads

Cryopreservation of eri and ailanthus silkworms using frozen gonads was investigated. First, we evaluated the freeze tolerance of ovary and testis in the eri silkworm, which showed high tolerance. Mating between frozen ovary-transplanted females and frozen testis-transplanted males produced 163.0 eggs, yielding 105.7 larvae per moth. In a second experiment, we tested the use of the eri silkworm as a host insect for gonad transplantation from ailanthus silkworm donors. A high success ratio for laid and hatched eggs was demonstrated for ovary transplantation (97.8 and 51.3 eggs per moth, respectively). For testis transplantation, however, the average number of hatched larvae was low (12.0). Mating between host eri females and males in which both frozen ovary and testis of the ailanthus silkworm had been transplanted produced 6.4 fertilized eggs per host moth. Our success in using cross subspecies cryopreservation between these wild silkworms could lead to the alternative use of hosts between species in other insects.     

Effect of incubation temperature after devitrification on quality parameters in human sperm cells

Sperm cryopreservation is common in assisted reproduction laboratories, providing a therapeutic option for several clinical conditions. This process has been optimized; however, the effect of post-thaw incubation temperature has been poorly studied. This work analyzed the effect of incubation temperature after devitrification on human sperm quality. Spermatozoa from normozoospermic donors were cryopreserved by vitrification. After devitrification, the spermatozoa were separated into two aliquots: (i) incubated at room temperature (RT, 22-25 °C) and (ii) incubated at 37 °C. Reactive oxygen species (ROS), viability, mitochondrial membrane potential (ΔΨM), phosphatidylserine externalization and motility were analyzed immediately after devitrification (control) and after 2, 4 and 6 h. Spermatozoa incubated at RT showed a conserved viability and ΔΨM compared to the control, while the incubation at 37 °C promoted a decrease in these parameters. The ROS levels were increased at both incubation conditions. The progressive motility was decreased in all experimental groups and the decrease was more pronounced under incubation at RT. No increase in phosphatidylserine externalization was observed. In conclusion, prior to use in assisted reproduction procedures, devitrified spermatozoa at RT conserve a better viability and ΔΨM than at 37 °C.     

Liver Freezing Response of the Freeze-Tolerant Wood Frog, Rana sylvatica, in the Presence and Absence of Glucose. II. Mathematical Modeling

The “two-step” low-temperature microscopy (equilibrium and dynamic) freezing methods and a differential scanning calorimetry (DSC) technique were used to assess the equilibrium and dynamic cell volumes in Rana sylvatica liver tissue during freezing, in Part I of this study. In this study, the experimentally determined dynamic water transport data are curve fit to a model of water transport using a standard Krogh cylinder geometry (Model 1) to predict the biophysical parameters of water transport: L_pg = 1.76 μm/min-atm and E_Lp = 75.5 kcal/mol for control liver cells and L_pg[cpa] = 1.18 μm/min-atm and E_Lp[cpa] = 69.0 kcal/mol for liver cells equilibrated with 0.4 M glucose. The DSC technique confirmed that R. sylvatica cells in control liver tissue do not dehydrate completely when cooled at 5°C/min but do so when cooled at 2°C/min. Cells also retained twice as much intracellular fluid in the presence of 0.4 M glucose than in control tissue when cooled at 5°C/min. The ability of R. sylvatica liver cells to retain water during fast cooling (≥5°C/min) appears to be primarily due to its liver tissue architecture and not to a dramatically lower permeability to water, in comparison to mammalian (rat) liver cells which do dehydrate completely when cooled at 5°C/min. A modified Krogh model (Model 2) was constructed to account for the cell–cell contact in frog liver architecture. Using the same biophysical permeability parameters obtained with Model 1, the modified Krogh model (Model 2) is used in this study to qualitatively explain the experimentally measured water retention in some cells during freezing on the basis of different volumetric responses by cells directly adjacent to vascular space versus cells at least one cell removed from the vascular space. However, at much slower cooling rates (1–2°C/h) experienced by the frog in nature, the deciding factor in water retention is the presence of glucose and the maintenance of a sufficiently high subzero temperature (≥−8°C).     

Presence of Platelet-Activating Factor and Its Receptor in Baboon (Papio spp) Spermatozoa

Platelet-activating factor [1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine; PAF] is a novel potent signaling phospholipid which has unique pleiotropic biological properties in addition to platelet activation. PAF has been detected in the spermatozoa in several species. The concentration of PAF is inversely related to human spermatozoal quality. PAF is present in squirrel monkey (a seasonal breeder) spermatozoa and is significantly higher during the breeding season. PAFs mechanism of action is a receptor-mediated event. There is no report on the presence of PAF or the PAF-receptor in nonhuman Old World primate spermatozoa. Therefore, the primary objective of this study was to determine if PAF is present in the spermatozoa from baboons, which are year-round breeders. A secondary objective was to determine the presence and localization of the PAF-receptor in spermatozoa. We extracted endogenous lipids from mature hybrid baboon (Papio spp) epididymal spermatozoa and assayed them for the presence of PAF by [ ¹²⁵ I]-radioimmunoassay. We also exposed baboon spermatozoa to PAF-receptor antibody followed by FITC-conjugated antibody. PAF was in all samples assayed (mean: 2.29 (±0.63) pmol/10 ⁶ spermatozoa). Baboon spermatozoa possess PAF-receptors most prevalently along the neck and midpiece regions. The data demonstrates that PAF and its receptor are present in baboon spermatozoa. Additional studies will elucidate the role of PAF in spermatozoal function.     

氧离子辐射对体外人精子自发化学发光、运动性、顶体反应和存活率的影响(英文)

我们研究了不同剂量(0,0.25,0.5,1,2,4,8,16,32,64Gy)3.17MeV/u氧离子对体外人精子自发化学发光(SCL)、运动性,顶体反应(AR)和存活率的影响。结果表明,精子SCL随辐射剂量明显增强,最低有效剂量为0.5Gy;精子运动性在0.5—2Gy,AR在0.5—4Gy辐射组均明显高于其对照组,但均随辐射剂量的进一步增加而明显降低;在0.25—8Gy剂量之间,精子存活率与其对照组相比无明显变化,但当剂量进一步增加至16—64Gy时,存活率急剧下降。这些结果提示,低剂量重离子辐射能对精子运动性及AR等功能产生兴奋效应。而大剂量则明显抑制这些功能,甚至造成精子死亡。其作用机制可能与重离子辐射诱导的自由基反应有关。     

精子为载体的基因转移研究

采用共培养和脂质体介导两种方法将金黄地鼠附睾内的精子与NFκB-luc+质粒共孵育后,再与成熟的卵母细胞进行体外受精,用PCR、Southern blot及荧光原位杂交(FISH)等技术检测外源基因在精子细胞和在受精卵中的存在及存在部位,并对相关数据进行统计学比较分析。结果表明,金黄地鼠附睾内的活精子具有主动捕获外源基因的能力,而且外源基因只存在于精子细胞的头部,而且通过受精将外源基因带入了卵细胞,脂质体的存在增加了外源基因转染精子的效率和转基因效率。Abstract: Incubate golden hamster spermatozoa and NFκB-luc+ in co-culture and liposome mediated methods, and then process the IVF with oocytes, And two groups of samples were detected by PCR, Southern Blot and FISH. Analyzed the relative data by statistics. The results showed that spermatozoa of Golden hamster epididymis can take the foreign DNA voluntarily, and the foreign DNA exist in the head of the spermatozoa can enter into zygotes by IVF. The exist of liposome can improve the foreign gene transfect ratio and transgenic ratio.     

Nuclear transfer of porcine embryos using cryopreserved delipated blastomeres as donor nuclei

Nuclear transfer protocol for the pig using cryopreserved delipated four- to eight-cell and morula stage embryos as nucleus donors was developed. Donor embryos, which had been delipated by micromanipulation following centrifugation for polarizing cytoplasmic lipid droplets, were cryopreserved with 1.5 M 1,2-propanediol and 0.1 M sucrose. Recipient cytoplasts were prepared from ovulated oocytes. Activation of oocytes could be induced more efficiently when electric stimulation was given 53 hr after the hCG injection or later (66–83%), compared with 52 hr or earlier (11–16%, P < 0.05), suggesting that aging after ovulation may be required for in vivo matured porcine oocytes to be activated by electric stimuli. Membrane fusion rates between donor blastomeres and enucleated oocytes were 88% (127/144) and 97% (56/58, P > 0.05) for the four- to eight-cell and morula stage embryos, respectively. In vitro developmental rates to the two-cell (53/100 vs. 35/65), four-cell (34/100 vs. 26/65), and morula stage (17/100 vs. 18/65) were the same between the nuclear transfer embryos with four- to eight-cell and morula nuclei. However, more embryos reconstituted with morula nuclei developed to blastocysts (15% vs. 6%, P < 0.05). These data demonstrated that blastomeres of cryopreserved, delipated porcine embryos can be used as donor nuclei for nuclear transfer. Frozen-thawed, delipated blastomeres can be efficiently isolated and fused, and therefore provide a useful source of donor nuclei. Mol. Reprod. Dev. 48:339–343, 1997. © 1997 Wiley-Liss, Inc.     

Atomic force microscopy and cytochemistry of chromatin from marsupial spermatozoa with special reference to Sminthopsis crassicaudata

Atomic force microscopy (AFM) of the nuclear topology of spermatozoa from two marsupial species, Sminthopsis crassicaudata and Trichosurus vulpecula was investigated to determine the structural organisation of the chromatin subunits. That of the former species is of special interest as it has a peripheral nucleohistone region (C2) as well as a nuclease-resistant, nucleoprotamine core region (C1). Atomic force microscopy showed that the C2 region contained clusters of 120–160 nm nodules, whereas the C1 region exhibited smaller 50–80 nm nodules. The spermatozoa nuclei of Trichosurus, which has mainly nucleoprotamines, contained higher order chromatin structures of similar size to those from the C1 region of Sminthopsis. This study shows that nucleohistones and nucleoprotamines of marsupial sperm form distinct higher order conformations. For the second part of this work, the chromatin density and affinity for cationic stains of Sminthopsis spermatozoa were determined. Spermatozoa were observed with the transmission electron microscopy (TEM) either unstained or stained with metal salts. In the unstained specimens, the C2 nucleohistone region appeared more electron-lucent than the C1 region. When large cations such as uranyl were used, the reverse situation was observed. Therefore, the electron-dense appearance of the C2 chromatin in conventionally stained material may be due to the presence of net negative DNA charges that attract the cations used for EM staining, whereas the C1 chromatin may lack excess DNA negative charges that attract these stains and thus appears less electron-dense. Mol. Reprod. Dev. 48:367–374, 1997. © 1997 Wiley-Liss, Inc.     

Effects of low temperatures on in vitro produced bovine zygotes

The objective of this research was to investigate the effects of cooling on the development of bovine zygotes. One-cell bovine embryos were maintained at 39°C (control), 20°C, 10°C, or 0°C for 5, 10, or 20 minutes, then cultured in vitro for 7 days and the proportion of embryos developing to the compact morula or blastocyst stage compared between different treatments. Duration of exposure time had no effect on development. Development rates to the compact morula or blastocyst stage were 3.9%, 11.4%, 17.4%, and 24.4% for zygotes maintained at 0°C, 10°C, 20°C, and 39°C, respectively, with differences in embryo yield between every treatment (P < 0.05). In a second experiment, bovine pronuclei (karyoplasts) and cytoplasts were cooled at 0°C or maintained at 39°C for 5 minutes. Pronuclear transplantation was then utilized to create 4 types of reconstructed embryos, those with: 1) non-cooled pronuclei and non-cooled cytoplasm, 2) non-cooled pronuclei and cooled cytoplasm, 3) cooled pronuclei and non-cooled cytoplasm, and 4) cooled pronuclei and cooled cytoplasm. The proportion of embryos developing to the blastocyst stage was highest when non-cooled pronuclei were transferred into non-cooled cytoplasm (18.9%), and similar to that of non-cooled, non-manipulated control zygotes (13.2%, P > 0.05). No embryos developed to the blastocyst stage when pronuclei (cooled or non-cooled) were transferred into cooled cytoplasm. However, zygotes with cooled pronuclei transferred into non-cooled cytoplasm yielded 4.5% blastocysts (P < 0.05). More embryos developed to the compact morula or blastocyst stage when non-cooled vs. cooled cytoplasm was utilized, regardless of whether the pronuclei were cooled (P < 0.05). These data demonstrate that pronuclei are more tolerant to low temperature exposure than is ovum cytoplasm. Mol. Reprod. Dev. 47:435–439, 1997. © 1997 Wiley-Liss, Inc.