Effect of beta-mercaptoethanol during in vitro fertilization procedures on sperm penetration into porcine oocytes and the early development in vitro

This study was carried out to determine the effects of beta-mercaptoethanol (bME) during a transient co-culture of gametes for 10 min, and/or the following culture until 6-9 h after insemination, on sperm penetration of porcine in vitro maturation (IVM) oocytes and the early development in vitro. When fresh spermatozoa were cultured in various concentrations of bME for 2 h, bME neutralized the stimulatory effect of caffeine-benzoate on sperm capacitation and the spontaneous acrosome reaction at 50-250 micromol/l. When 50 micromol/l bME were added during a transient co-culture of gametes for 10 min, the sperm penetration rate was reduced 9 h after insemination (70.5-82.0% vs 90.5-94.0% in the absence of bME), but the incidence of monospermic penetration was not affected. When 50 micromol/l bME were supplemented during culture after a transient co-culture, the sperm penetration rate was not affected, but the incidence of monospermy oocytes was increased (43.9-45.8% vs 31.7-34.3% in the absence of bME). The presence of bME following a transient co-culture minimized a decrease of oocyte glutathione content at 6 h after insemination (7.9 pmol/oocyte before in vitro fertilization (IVF), 6.7 pmol/oocyte in the presence of bME vs 5.5 pmol/oocyte in the absence of bME). When the distribution of cortical granules was evaluated 1 h after activation with calcium ionophore, mean pixel intensity of fluorescein isothiocyanate-labeled peanut agglutinin (FITC-PNA) at the cortex region was lower in the oocytes activated and cultured in the presence of 50 micromol/l bME. Although the presence of 50 micromol/l bME during a transient co-culture for 10 min and the following culture did not increased blastocyst formation (29.6-37.7%), 50 micromol/l bME during the following culture significantly increased the mean cell numbers per blastocyst (73.3-76.4 vs 51.2 in the presence and absence of bME respectively). These results demonstrate that supplementation with bME during IVF procedures, except during a transient co-culture period of gametes in the presence of caffeine, has a beneficial effect in maintaining the function of gametes, the incidence of normal fertilization and, consequently, the quality of IVF embryos.     

Replacement of nuclear protein by histone in pig sperm nuclei during in vitro fertilization

Sperm-specific nuclear protamines are dissociated before decondensation of sperm nuclei during fertilization in pigs. In the present study, replacement of nuclear protein by histone in boar spermatozoa during in vitro fertilization was evaluated by immunohistochemistry using anti-histone antibody. First, the specificity of the antibody used in this study was examined. Immunohistochemistry of the testes and epididymides indicated that somatic nuclei, but not elongated spermatids or maturing spermatozoa, were immunoreactive. Furthermore, immunoreaction was diminished after the antibody had been preincubated with unfractionated histone, indicating that the antibody was specific for the somatic nuclear histone. Immunohistochemistry of serial sections of oocytes, which were matured and co-cultured with boar spermatozoa for 2 to 6 h indicated that, at 2 to 3 h after insemination, penetrating sperm nuclei in the condensed state were not immunoreactive. At 4 to 5 h after insemination, some of the condensed sperm nuclei were immunoreactive in part or over the whole area of the nucleus, and all of the decondensing nuclei and male pronuclei were immunoreactive. At 6 h after insemination, the decondensing sperm nuclei and well-developed male pronuclei were immunoreactive. These results imply that, in pigs, remodelling of sperm nuclear protein from protamine to histone is initiated at the time of sperm penetration, before onset of decondensation and male pronuclear formation.     

Evaluation of calcium-free Tyrode's sperm capacitation medium for use in bovine in vitro fertilization

Our objective was to determine if a bovine sperm capacitation technique, developed with zona-free hamster oocytes, could be used for the in vitro fertilization of in vitro matured bovine zona-intact oocytes. Bovine cumulus-enclosed primary oocytes from 2- to 5-mm follicles were matured in tissue culture Medium 199 containing Earle's salts and bicarbonate and supplemented with 10% fetal calf serum, FSH (10 micrograms/ml), and estradiol-17 beta (1.5 microgram/ml) for 24 h at 37 degrees C under paraffin oil. Ejaculated bovine sperm, washed thrice in bovine serum albumin-saline (pH 7.6) and capacitated for 4 h in Ca(++)-free Tyrode's medium (pH 7.6), were diluted to 2 x 10(6) sperm/ml in Medium 199 supplemented with 10% fetal calf serum. Oocytes were added (10/500 microliters droplet) to this medium containing the capacitated sperm, freeze-thawed killed sperm, or no sperm and incubated for 8 h before transfer to fresh medium and then incubated for 40 h. At the end of each incubation, a portion of the oocytes were stained and evaluated for development or fertilization. After 24 h of culture, 49% of the oocytes had matured (metaphase II). Fertilization rates were 55.6% after exposure of all oocytes to Ca(++)-free Tyrode's capacitated sperm and 82.5% if only metaphase II oocytes were selected. The parthenogenetic controls were negative (1.4% and 0%). Therefore, the Ca(++)-free Tyrode's sperm capacitation technique can be used for bovine in vitro fertilization studies.     

Evaluation of a TEST-yolk sperm capacitation system for use in bovine in vitro fertilization.

Bovine sperm acquire the ability to penetrate zona-free hamster oocytes (capacitation) after incubation in TEST-yolk buffer. Our objective was to determine whether such sperm could penetrate zona-intact bovine oocytes in vitro. Bovine cumulus enclosed oocytes from 2- to 5-mm follicles were incubated in maturation medium for 24 h at 37 degrees C. Ejaculated bovine semen was diluted 1: 10 in TEST-yolk buffer, cooled to 4 degrees C, and stored for 8 h to induce capacitation. Sperm were then washed thrice in pH 7.6, .15 M NaCl containing .1% bovine serum albumin V (37 degrees C) and diluted to 2 x 10(6) sperm/ml in fertilization medium. Droplets of fertilization medium containing capacitated sperm, killed sperm, or no sperm were made under paraffin oil. Oocytes (matured 24 h) were added and cocultured with sperm for 8 h and then transferred to fresh fertilization medium for 40 h. After 24 h, 53% of the oocytes had matured (metaphase II). The fertilization rate of the metaphase II oocytes (203) with TEST-yolk capacitated sperm was 87%, whereas the parthenogenetic controls were 2 and 0%, respectively. Therefore, TEST-yolk buffer can be used to capacitate bull sperm for in vitro fertilization.     

Effects of cryopreservation on sperm quality, nuclear DNA integrity, in vitro fertilization, and in vitro embryo development in the mouse

Efficient freezing, archiving, and thawing of sperm are essential techniques to support large scale research programs using mouse models of human disease. The purpose of this study was to investigate the effects of variable combinations and concentrations of cryoprotectants on sperm-assessment parameters of frozen-thawed mouse sperm in order to optimize cryopreservation protocols. Sperm was frozen using combinations of 3% skim milk + 0.2 or 0.3 M nonpermeating raffinose with either permeating glucose, fructose, propylene glycol, ethylene glycol, glycerol, or sodium pyruvate in CD-1, C3FeB6F1/J, B6129SF1, C57BL/6NCrIBR, 129S/SvPaslco, and DBA/2NCrIBR mice. Sperm-assessment parameters included progressive motility, plasma membrane integrity (SYBR-14 + PI), in vitro fertilization rate, and in vitro embryo development rate to blastocyst. DNA content analysis of sperm was measured by the sperm chromatin structure assay (SCSA). 0.3 M raffinose with 0.1 M fructose significantly improved post-thaw sperm-assessment parameters for CD-1, C3B6F1, B6129SF1 mice (P < 0.05-0.01), whereas 0.2 M raffinose with 0.1 M glycerol or 0.1 M fructose enhanced sperm assessment values for C57BL/6 and 129S mice (P < 0.01), compared to 0.3 M raffinose alone. DNA fragmentation during cryopreservation was significantly increased in all strains evaluated when compared with fresh control sperm in a strain-dependent manner (P < 0.01). Supplementation with permeating glycerol or fructose to the cryoprotectant (CPA) solution showed a significant protective effect to DNA integrity when cryopreserving sperm from C57BL/6 and 129S mice. Damage to sperm DNA significantly decreased the rate of in vitro embryo development to blastocyst in C57BL/6 mice. The type of monosaccharide sugar or polyols, CPA molarity, and combination of permeating and nonpermeating cryoprotectant are significant factors for improving progressive motility, plasma membrane integrity, DNA integrity, in vitro fertilization rate, and in vitro embryo development rate to blastocyst in cryopreserved mouse sperm.     

Effect of in vitro fertilization on gene expression and development of mouse preimplantation embryos

In vitro culture (IVC) of preimplantation mouse embryos is associated with changes in gene expression. It is however, not known if the method of fertilization affects the global pattern of gene expression. We compared gene expression and development of mouse blastocysts produced by in vitro fertilization (IVF) versus blastocysts fertilized in vivo and cultured in vitro from the zygote stage (IVC) versus control blastocysts flushed out of the uterus on post coital day 3.5. The global pattern of gene expression was assessed using the Affymetrix 430 2.0 chip. It appears that each method of fertilization has a unique pattern of gene expression and development. Embryos cultured in vitro had a reduction in the number of trophoblastic cells (IVF 33.5 cells, IVC 39.9 cells, and 49.6 cells in the in vivo group) and, to a lesser degree, of inner cell mass cells (12.8, 11.7, and 13.8 respectively). The inner cell mass nuclei were larger after culture in vitro (140 microm(2), 113 microm(2), and 86 microm(2) respectively). Although a high number of genes (1912) was statistically different in the IVF cohort when compared with the in vivo control embryos, the magnitude of the changes in gene expression were low and only a minority of genes (29 genes) was changed more than fourfold. Surprisingly, IVF embryos were different from IVC embryos (3058 genes were statistically different, but only three changed more than fourfold). Proliferation, apoptosis, and morphogenetic pathways are the most common pathways altered after IVC. Overall, IVF and embryo culture have a profound effect on gene expression pattern and phenotype of mouse preimplantation embryos.     

Cytoskeleton localization in the sperm head prior to fertilization

Three major cytoskeletal proteins, actin, tubulin and spectrin, are present in the head of mammalian spermatozoa. Although cytoskeletal proteins are implicated in the regulation of capacitation and the acrosome reaction (AR), their exact role remains poorly understood. The aim of this study was to compare the distribution of the sperm head cytoskeleton before and after the AR in spermatozoa representing a range of acrosome size and shape. Spermatozoa from the human and three rodents (rat, hamster and grey squirrel) were fixed before and after the AR in appropriate medium in vitro. Indirect immunofluorescent localization of cytoskeletal proteins was undertaken with antibodies recognizing actin, spectrin and alpha-tubulin. Preparations were counterstained with propidium iodide and examined by epifluorescent and confocal microscopy. Our results clearly demonstrated changes in localization of cytoskeleton during the AR, mainly in the apical acrosome with further changes to the equatorial segment and post-acrosomal regions. The pattern of cytoskeletal proteins in the sperm head of all the species was similar in respect to various sub-compartments. These observations indicated that the sperm head cortical cytoskeleton exhibits significant changes during the AR and, therefore, support the image of cytoskeletal proteins as highly dynamic structures participating actively in processes prior to fertilization.     

Early history of in vitro fertilization

Although in vitro fertilization (IVF) is used widely for a variety of purposes, it is often not appreciated how this technology was developed. A large number of experiments beginning in 1878 contributed to the first successful reports of IVF over 75 years later. The discovery of sperm capacitation in 1951 was central to the development of IVF technology, and it was rapidly followed by the first convincing reports of IVF in several species. The ability to fertilize oocytes in vitro has allowed major advances to be made into understanding the mechanisms involved in fertilization and early development, and IVF now supports reproductive biotechnology in animals and in humans. This article is a historical review of key experiments that helped to provide the basis for present day IVF procedures, placed into context with current practice.     

Increased disruption of sperm plasma membrane at sperm immobilization promotes dissociation of perinuclear theca from sperm chromatin after intracytoplasmic sperm injection in pigs

The effects of sperm-immobilization methods on decondensation of sperm chromatin and retention of subacrosomal sperm perinuclear theca (SAR-PT) after intracytoplasmic sperm injection (ICSI) were examined in pigs. Sperm membrane damage caused by different immobilization methods by rubbing with a micropipette without piezo pulses (R), or with a low (L) or high (H) intensity of piezo pulses while rubbing, was assessed by the time required for staining of sperm heads with eosin Y solution. The average time for staining of sperm heads immobilized by the R, L or H treatments was 76, 41 or 26 s, respectively. The fertilization rate following ICSI was increased by sperm immobilization by piezo pulses compared with R, but increased intensity of pulses from L to H did not cause further improvements (29, 48 and 47%, respectively). An immunofluorescence study revealed that H immobilization promoted the dissociation of SAR-PT from sperm chromatin compared with L and R, and it increased the frequency of male pronuclear formation in which chromatin appeared uniformly decondensed. With in vitro fertilization (IVF), SAR-PT disassembled coordinately with sperm chromatin decondensation and it was not detectable around male pronuclei. This was different from most of the oocytes after ICSI in which remnants SAR-PT were detected adjacent to male pronuclei. We concluded that increased damage on the sperm plasma membrane at immobilization improved fertilization rates and decondensation of sperm chromatin after ICSI due to the accelerated dissociation of SAR-PT from the sperm nucleus. Also, the behavior of SAR-PT after ICSI was different from that observed in oocytes after IVF.     

Effect of preparation method on the glycaemic index of novel potato clones

The purpose of this study was to investigate whether the effects of cooling and reheating on the glycaemic index (GI) of novel potato clones (selections) differed depending on selection and whether cooling altered starch absorption in vivo. We conducted 3 experiments using 4 novel potato clones in healthy subjects. Experiment 1: the GI of 4 selections each prepared in 3 ways (freshly boiled, cooled, or cooled and reheated) was measured in 2 groups of 10 subjects (each group tested 2 selections). Experiment 2 (n=10): two selections from Experiment 1 were re-tested one year later, by a different subject group. Experiment 3 (n=10): two selections from Experiment 1 were tested by subjects from Experiment 2 to assess the rate and extent of starch absorption using the second-meal effect and the breath hydrogen method, respectively. Experiment 1 demonstrated a selection×treatment interaction for GI (p=0.024); cooling reduced the GI of two selections by 40-50% (p<0.05) but reduced GI of the other 2 by only 8-10% (ns). Experiment 2 confirmed the selection×treatment interaction (p=0.018) seen in Experiment 1. Experiment 3: cooling reduced the GI by an average of 37% (p<0.05) but only increased starch malabsorption in vivo from 3% to 5% (p=0.021); there was no significant second-meal effect. It is concluded that the effect of cooling on the GI of potatoes may vary from 0-50% depending on selection. However, the mechanism for the effect is not clear: the 2% increase in starch malabsorption seen upon cooling potatoes was not nearly enough to account for the 37% reduction in GI.     

Effect of time of insemination on number of accessory sperm, fertilization rate, and embryo quality in nonlactating dairy cattle.

Two experiments were conducted to determine the effect of insemination time on number of accessory sperm per embryo (ovum), fertilization rate, and embryo quality. Semen was collected from three fertile Holstein bulls and cryopreserved in egg yolk-citrate-glycerol. In experiment 1, cows were continuously monitored for behavioral estrus by the HeatWatch estrous detection system and were artificially inseminated (AI) with one 0.5-ml straw (25 x 10(6) sperm) at the onset of estrus (AI 0 h), 12 h after onset (AI 12 h), or received natural service at 0 h (Nat 0 h) from one of three bulls. From 150 inseminations, 115 embryos and ova (AI 0 h: n = 39; AI 12 h: n = 39; Nat 0 h: n = 37) were recovered 6 or 7 d after insemination. Fertilization rates differed between treatments (AI 0 h: 67%; AI 12 h: 79%; Nat 0 h: 98%). Median accessory sperm per embryo (ovum) also differed (AI 0 h: 1; AI 12 h: 10; and Nat 0 h: 27) and paralleled the fertilization rate. Embryo quality was not affected by insemination time or natural service. In experiment 2, cows received AI at 0, 12, or 24 h (AI 24 h) after the onset of estrus as determined by HeatWatch. From 154 inseminations, 117 embryos and ova (AI 0 h: n = 39; AI 12 h: n = 39; AI 24 h: n = 39) were recovered 6 or 7 d after insemination. Fertilization rates did not differ in experiment 2 (AI 0 h: 66%; AI 12 h: 74%; AI 24 h: 82%); however, a trend toward a higher fertilization rate accompanied AI 24 h. Median accessory sperm values increased from AI 0 h (1) to AI 24 h (4). Embryo quality declined with AI at increasing intervals after onset of estrus, as percentages of excellent and good, fair and poor, and degenerate embryos were as follows: 77, 15, 8; 52, 38, 10; and 47, 19, 34 for the 0-, 12-, and 24-h inseminations, respectively. Results indicate AI 12 h after the onset of estrus provides a compromise between potential fertilization failure (AI 0 h) and embryo failure (AI 24 h), despite increased accessory sperm per embryo (ovum) after AI 24 h. Artificial insemination 12 h after onset of estrus should optimize fertility of dairy cattle through an acceptable fertilization rate, number of accessory sperm per embryo, and desirable embryo quality.     

Early in vitro fertilization improves development of bovine ova heat stressed during in vitro maturation

The objectives were to examine the development of embryos derived from control (38.5°C) or heat-stressed ova [41.0°C during the first 12 h of in vitro maturation (hIVM)] when in vitro fertilization (IVF) was performed at 16, 18, 20, 24, or 30 hIVM. Effects of heat stress in compromising ovum development depended on when IVF was performed (in vitro maturation temperature × IVF time interaction). When IVF was performed at 24 or 30 hIVM, fewer heat-stressed ova developed to the blastocyst stage compared with the respective controls. In contrast, when IVF was performed at 16, 18, or 20 hIVM, more heat-stressed ova developed to the blastocyst stage compared with the respective controls. Performing IVF earlier than usual was beneficial, because the ability of heat-stressed ova to develop to the blastocyst stage was improved when IVF was performed at 18 or 20 vs. 24 hIVM. Blastocyst stage and quality were equivalent to non-heat-stressed controls regardless of IVF time. Control ova undergoing IVF at 20, 24, 30, or 32 hIVM and heat-stressed ova undergoing IVF at 16, 18, 20, or 24 hIVM were compared for blastocyst development by multisource regression. Although linear and quadratic slopes were similar, heat stress reduced the peak and shifted the developmental response of ova by 7.3 h. In other words, obtaining optimal blastocyst development from heat-stressed ova would depend on performing IVF at 19.5 hIVM compared with 26.7 hIVM for non-heat-stressed controls. Heat-induced reductions in peak blastocyst development significantly reduced the window of time available to perform IVF and obtain ≥20% blastocyst development. In summary, results support an effect of heat stress to hasten developmentally important events during oocyte maturation. The inability of earlier IVF to fully restore the development of heat-stressed ova to that of non-heat-stressed controls highlights the importance of further study.     

纺丝液配备方法对PS/PVP纳米纤维膜性能的影响

采用特殊的纺丝液配备方法,依靠新型的气泡静电纺丝技术,成功纺制出PS/PVP纳米纤维膜,并对纳米纤维膜的微观形态、亲水性能、强伸性能、平均孔径及空气流速进行测试与分析。结果表明:不同配备方法的纺丝液获得的纳米纤维膜的微观形态、亲水性能、强伸性能、平均孔径及空气流速不同。表面粗糙程度高、孔洞多、有凹槽的纳米纤维,其形成的纳米纤维膜的亲水性能优,断裂强度高,断裂伸长率低,平均孔径和空气流速增加。     

Selective sperm binding to pig oviductal epithelium in vitro

The sperm reservoir in the caudal isthmus of the oviduct of a number of species is created by binding of spermatozoa to oviductal epithelium. The sperm reservoir fulfills a number of functions such as control of sperm transport, maintenance of sperm viability and modulation of capacitation. The initial capacities of ejaculated and epididymal boar spermatozoa to bind to oviductal epithelium were investigated using a modified pig oviductal explant assay. The number of spermatozoa that bound to 0.01 mm(2) of explant surface was used as the parameter of binding capacity. Binding of spermatozoa to oviductal epithelial explants was dependent in a linear manner on the number of spermatozoa added (P < or = 0.05). No difference was found in initial sperm binding between isthmic and ampullar explants. There was no effect of the stage of the oestrous cycle or the reproductive status of the female donor. There was a significant effect (P < or = 0.05) of the individual boar on the binding index. The binding index correlated negatively with the percentage of spermatozoa with cytoplasmic droplets and the percentage of morphologically abnormal spermatozoa (P < or = 0.05). Epididymal spermatozoa showed significantly lower initial binding capability than did ejaculated spermatozoa from the same boars (P < or = 0.05); therefore, components of seminal plasma may play a role in the binding process. The individual differences revealed by this study and their relation to morphology and contact of spermatozoa with seminal fluid indicate a selective function of sperm-oviduct binding.     

Role of myometrial activity in sperm transport through the genital tract and in fertilization in sows

The effects of stimulation and suppression of uterine contractility at about the time of insemination on sperm distribution and fertilization in multiparous sows are described. For assessment of fertilization, sows were inseminated about 28 h before (synchronized) ovulation and killed at day 5 after ovulation (n = 53). For assessment of sperm distribution, sows were inseminated about 20 h before expected ovulation and were killed 12 h later (n = 26). At 10 min before insemination, sows received an intrauterine infusion of one of three solutions: (i) saline (control); (ii) 0.60 mg clenbuterol hydrochloride to suppress contractility; or (iii) 1 mg cloprostenol to stimulate contractility. Both clenbuterol and cloprostenol reduced median fertilization rate (P < 0.05) and median number of accessory sperm cells (P < 0.05). Distribution of sperm cells was also affected by treatments. Clenbuterol increased, and cloprostenol decreased, the number of sperm cells (P < 0.05) in the proximal 20 cm of the uterine horn and in the uterotubal junction. In addition, clenbuterol tended to increase and cloprostenol tended to decrease the number of sperm cells in the isthmus, although these effects were not significant. However, relative to the number of sperm cells in the uterus, clenbuterol treatment reduced the number of sperm cells in the uterotubal junction and oviduct, in contrast to cloprostenol. Cloprostenol increased the reflux of semen during insemination. It is hypothesized that suppression of uterine contractility increases transuterine transport time, reducing the ability of sperm cells to enter the uterotubal junction and the oviduct. Stimulation of uterine contractility above a certain level probably increases reflux and impedes transuterine transport of sufficient numbers of sperm cells.     

Individual variation for in vitro fertilization success in dairy bulls

Individual semen samples from 29 bulls in routine artificial breeding service were tested for their capability to achieve in vitro fertilization. Ejaculated semen was diluted, .1 ml of semen in 2 ml of a modified Tyrode's medium with an osmolality of 340 mOsmol/kg, washed thrice, incubated 3 h at 37 degrees C before being used for in vitro fertilization, or incubated 4 h and 8 h before assessment of motility, capacitation, and acrosome integrity. The degree of variability in percentage of oocytes fertilized was assessed along with several factors that might contribute to this variation. Variation among bulls was not significantly different. Variation from one replicate to another was high. Variation was found in motility, capacitation, and frequency of acrosome reaction, but these variables were not significantly correlated to fertilization rate in vitro.     

TiO2溶胶制备工艺对直接染料Sol-Gel法固色影响

以钛酸四丁酯作为前驱物制备溶胶,并将制备的溶胶应用到直接染料染色的棉机织物的固色,探讨了溶胶制备工艺对固色效果的影响.得到制备溶胶的优化工艺:钛酸四丁酯22 mL,乙醇60mL,抑制剂冰醋酸12 mL,去离子水2 mL,冰醋酸调pH至5,30℃搅拌1.5 h.该条件下制备的溶胶能达到较理想的固色效果,其中耐洗色牢度中的棉布沾色牢度提高1.5级,湿摩擦牢度提高1级.     

Effect of osmolality and glycosaminoglycans on motility, capacitation, acrosome reaction, and in vitro fertilizability of bovine ejaculated sperm

Bovine ejaculated semen was placed in a modified Tyrode's medium with albumin, lactate, and pyruvate. The sperm were washed three times and subjected to nine treatment in a 3 X 3 factorial arrangement. Treatments consisted of osmolality (exposure to 380 mOsmol/kg medium for 5 min, exposure to 340 or 295 mOsmol/kg medium for the entire incubation period), and the presence or absence of glycosaminoglycans (100 micrograms/ml chondroitin sulfate A or 10 micrograms/ml heparin). Sperm were examined at 4.5 h, 8 to 9 h, and 24 to 25 h of incubation (37 degrees C, 5% CO2, and 95% air). Heparin caused head-to-head agglutination of sperm, raised the percent sperm without seminal antigens over the acrosome (capacitated) by 20% at 4.5 h, and doubled the percent of acrosome-reacted sperm. However, this stimulation did not improve in vitro fertilizability. Chondroitin sulfate A tended to maintain motility, but did not affect capacitation or the acrosome reaction, possibly due to glucose inhibition. Both high osmolality treatments tended to reduce motility, especially after 24 h of incubation when the 340 osmolality treatment reduced motility by 14% over the 295 treatment. No consistent effect on capacitation was observed. The 340 and 380 osmolality treatments induced 8.6 and 6.1% more acrosome reactions by 24 h than the 295 treatment. The 340 mOsmol/kg treatment yielded insignificantly higher in vitro fertilization rates, as evidenced by development of zygotes to the two-cell stage. Lack of statistical significance was due to high variation with in vitro fertilization rates.     

叶黄素-花青素脂质体的制备及其体外抗氧化活性研究

采用分段乳化结合超声薄膜法制备氧化性更强、更稳定的叶黄素–花青素脂质体。结果表明:叶黄素的乳化最佳HLB值为10.9,选用的复合表面活性剂双乙酰酒石酸单甘油酯和辛奎酸甘油酯为42∶58。叶黄素与花青素在60℃,p H 9,膜材用量为7.9%的条件下,超声乳化47 min,包封率可达38%;体外抗氧化实验(ABTS自由基、DPPH自由基和OH·清除实验)表明叶黄素和花青素具有协同抗氧化作用。     

Signal transduction mechanisms involved in in vitro ram sperm capacitation

We validate the chlortetracycline (CTC) technique for the evaluation of capacitation and acrosome reaction-like changes in ram sperm, carrying out a double estimation of the acrosome status after treatment with lysophosphatidylcholine, using fluorescein isocyanate (FITC)-RCA/ethidium homodimer 1 (EthD-1) and CTC/EthD-1. Highly consistent results and a positive correlation between the results of acrosome-reacted sperm evaluated with both techniques were obtained. In this study, we evaluate the effects of ram sperm capacitation of BSA, Ca(2+), NaHCO(3) and cAMP agonists and their influence on the associated protein tyrosine phosphorylation. We found a time-dependent increase in capacitation related to protein tyrosine phosphorylation, either in the absence or the presence of BSA. The addition of an increasing concentration of cholesterol to samples containing BSA did not influence results. The effect of bicarbonate was concentration-dependent, with a significantly lowered value of non-capacitated sperm in the presence 18 and 25 mM. The addition of extracellular calcium did not significantly increase either the proportion of capacitated sperm or the protein tyrosine phosphorylation signalling, although a significantly higher value of acrosome-reacted sperm was found in samples containing 4 mM Ca(2+). cAMP agonists increased capacitated sperm and protein tyrosine phosphorylation signalling. The inhibition of protein kinase A by H-89 caused a decrease in sperm capacitation. Addition of a calcium-entry blocker (Verapamil; Sigma) did not influence results, which suggests that the calcium entry blocker was unable to inhibit the calcium influx associated with capacitation in ram sperm. Our findings might benefit our understanding of the biochemical mechanisms involved in mammalian sperm capacitation and ultimately, fertility.