酪氨酸磷酸化在精子获能过程中的作用

在精子细胞发生顶体反应并使卵母细胞受精之前,获能是一个重要的生理先决条件。获能是精子在雌性生殖道中进一步成熟的复杂现象,其赋予精子以增强活性的能力,使精子能够与卵母细胞透明带（ZP）相互作用,进行顶体反应并与卵母细胞质膜融合,进而完成受精过程。然而精子获能的分子机制十分复杂,目前还未完全明确,但获能后的精子会有诸多结构及生化方面的变化,如蛋白酪氨酸磷酸化、精子膜胆固醇外流、活性氧的产生及精子膜超极化。蛋白质通过磷酸化或去磷酸化调节精子获能和顶体反应等一些重要的现象,这是精子到达、结合、穿透和融合卵母细胞所必需的过程。因此蛋白磷酸化是获能的一个非常重要的过程,尤其是在酪氨酸残基处的磷酸化是获能过程中发生的最重要的事件之一,且酪氨酸磷酸化可能是细胞中信号转导途径的主要甚至是唯一的指标。作者主要针对蛋白磷酸化、精子中酪氨酸磷酸化的发现、作用和定位,以及影响获能过程中酪氨酸磷酸化的几个因素进行阐述。     

硫酸铜对泥鳅精子活力的影响

以泥鳅精子为受试材料,应用计算机辅助精子分析系统,研究硫酸铜(0.1、1、10 mg/L)对泥鳅的精子活力的影响.结果表明,在0 h试验组,0.1 mg/L的硫酸铜明显降低泥鳅精子运动速度,1 mg/L的硫酸铜对运动时间和速度产生显著抑制作用,10 mg/L的硫酸铜时运动百分数、运动时间和速度都产生明显影响(P<0.0...     

动物精液品质评定方法的研究进展

人工授精和体外受精技术的广泛应用,使精液保存技术得到了快速发展。精子的质量和功能状态直接影响其受精能力。文章综合阐述了近几年动物精液品质评定的方法:精子质膜完整性检测、线粒体活性的测定、受精能力的检测和精液中酶活性的测定。     

Association of SNPs in GnRH gene with sperm quality traits of Chinese water buffalo

Hypothalamic gonadotropin-releasing hormone (GnRH) controls the activity of hypothalamic–pituitary–gonadal axis and plays a key role in the reproductive performance of animals. In this study, five single nucleotide polymorphisms (SNPs), namely g.991T > C, g.1041T > C g.3424T > C, g.3462C > A and g.3463Inde A, were detected in the GnRH gene of 162 water buffaloes by Sanger sequencing. Each SNP was associated with more than two sperm quality traits of ejaculate volume, sperm concentration, post-thaw sperm motility and sperm abnormality. g.3424T > C and g.3462C > A were related to these four traits and had a remarkable effect on ejaculate volume. The three other SNPs were related to sperm concentration, post-thaw sperm motility and sperm abnormality. Moreover, six haplotypes (H1: TCCAI, H2: CTTC-, H3: TCCCI, H4: CTTA-, H5: CCTA- and H6: CTCC-) composed of five SNPs comprising seven different combined genotypes were generated by linkage disequilibrium analysis. Statistics followed by one-way ANOVA indicated that water buffaloes with the haplotype combination H1H1 had the highest genotypic frequency, and those with the H4H4 haplotype combination had the highest ejaculate volume. The sperm concentration of those with haplotype combination H1H5 was higher than that of the other genotypes. In summary, our study showed a remarkable association between the SNPs of GnRH and sperm quality traits of Chinese water buffalo.     

Recovery of sperms bearing X chromosomes with different concentrations of magnetic nanoparticles in ram

Pre-conceptual sex selection is still a highly debatable process whereby X and Y chromosome bearing spermatozoa are isolated before oocyte fertilization. Recently, magnetic nanoparticles (MNP) have been used to determine X and Y chromosomes bearing spermatozoa as a result of searching for a cheap, highly efficient method using non-toxic materials. This study aimed to recover the sperm bearing X chromosomes in ram with different concentrations of MNP and then evaluate the success of this method using polymerase chain reaction (PCR). Ram sperms were divided into four groups, treated with 0 (control), 50, 100 and 200 μg/ml MNP, respectively. MNP was used to restore sperm cells bearing X chromosomes. Upon recovery, the PCR was performed to identify the X and Y sperms, Methyl ThiazoleTetrazolium (MTT), to assess MNP toxicity and sperm viability and acridine orange (AO) to evaluate sperm DNA integrity. The results of PCR revealed that the treatment of spermatozoa- bearing X chromosomes with 50 μg/ml MNP had the highest effects on the recovery of X sperm rather than the other concentrations of MNP. However, the concentrations of MNP did not have any toxic effects on spermatozoa, sperm viability and, DNA integrity, but the high concentration of MNP (200 μg/ml) significantly reduced DNA integrity. According to MTT and AO results, the concentrations of MNP used in this study had no toxic effects on spermatozoa and did not reduce the sperm viability and DNA integrity, except that 200 μg/ml MNP significantly reduced DNA integrity.     

日粮粗蛋白质及能量水平对架子牛肥育效果影响的研究

试验采用2～3岁,体重在305～350kg的架子牛,按高蛋白高能量(精料1号)、高蛋白低能量(精料2号)、低蛋白高能量(精料3号)、低蛋白低能量(精料4号)四组日粮进行为期90d的肥育。试验结果表明,当架子牛体重在320～420kg的情况下,高蛋白高能量日粮组日增重明显高于其他三组(P<0.01)。当体重超过420kg以上时,高蛋白高能量组日增重与低蛋白高能量组比较差异显著(P<0.05),与其他两组比较差异极显著(P<0.01)。从经济效益上分析,由高到低,依次为高蛋白高能量、低蛋白高能量、高蛋白低能量、低蛋白低能量。结论:架子牛肥育前期日粮(按风干计)的粗蛋白质水平应达到13.5%～14.0%,消化能在11.60～11.70MJ/kg;肥育后期粗蛋白质水平应达到12%,消化能要在11.80MJ/kg以上。     

Is sperm cryopreservation in absence of permeable cryoprotectants suitable for subfertile donkeys?

Sperm from fertile donkeys have been successfully frozen in absence of permeable cryoprotectants. The aim of this study was to determine whether this cryopreservation method is suitable for subfertile donkeys in comparison to conventional sperm freezing with glycerol. Ejaculates were collected from four Andalusian Donkeys: three fertile and one subfertile. Semen was frozen with an extender containing glycerol (GLY), or adding instead sucrose 0.25 molar and 1% bovine serum albumin (SUC) as non‐permeable cryoprotectants. After thawing, samples were assessed for total (TM, %) and progressive (PM, %) sperm motility by CASA, plasma membrane integrity (PMI, %) by epifluorescence microscopy and DNA integrity (DFI, %) by SCSA. Results (mean ± SD) were compared between extenders in fertile and subfertile donkeys using the Student's t test. No differences between GLY and SUC treatments were found in the fertile group for the sperm parameters assessed. In subfertile donkey ejaculates, GLY resulted in significantly higher values than SUC for TM (25.5 ± 3.1 vs. 19.6 ± 1.9) and PM (13.3 ± 5.1 vs. 4.0 ± 1.2), respectively. In conclusion, considering all the sperm parameters assessed, sperm freezing in absence of permeable cryoprotectants may not be still an option for cryopreservation of subfertile donkey sperm.     

Nitric oxide impacts bovine sperm capacitation in a cGMP‐dependent and cGMP‐independent manner

We aimed to elucidate whether NO acts in in vitro sperm capacitation in bovine via cGMP/PKG1 pathway. For this, cryopreserved bovine sperm were capacitated in vitro with 20 µg/ml heparin (Control) plus treatments: 1 mM L‐arginine (L‐arg, NO precursor), 50 µM Rp‐8‐Bromo‐β‐phenyl‐1,N²‐ethenoguanosine‐3′,5′‐cyclic monophosphorothioate (Rp‐8‐Br‐cGMPS, selective inhibitor of the binding site for cGMP in PKG1), 1 mM 2‐Phenyl‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl 3‐oxide (PTIO, NO scavenger), and the combinations of L‐arg + RP‐8‐Br‐cGMPS and L‐arg + PTIO. Sperm motility and vigour were determined by phase‐contrast microscopy, capacitation status by chlortetracycline staining, and the intracellular concentration of cGMP was measured by ELISA. Data were subjected to analysis of variance and means compared with SNK test at 5% probability. Motility and vigour were lower in sperm treated with PTIO when compared to Control and other treatments (p < .05). The L‐arg treatment showed the highest percentage of capacitated sperm when compared to the Control and other treatments (Rp‐8‐Br‐cGMPS, L‐arg + Rp‐8‐Br‐cGMPS and PTIO) (69.8 ± 3.4%, 51.2 ± 3.0, 51.1 ± 2.1, 51.2 ± 3.0 and 45.5 ± 2.7, respectively) (p < .05). The capacitation ratio (%) was lower in treatments with Rp‐8‐Br‐cGMPS, L‐arg + Rp‐8‐Br‐cGMPS and PTIO, respectively (p < .05). Lastly, cGMP concentration (pmol/ml) was lower in PTIO and L‐arg + PTIO (1.3 ± 0.3 and 1.6 ± 0.4) and was higher in Rp‐8‐Br‐cGMPS and L‐arg + Rp‐8‐Br‐cGMPS (3.7 ± 0.4 and 4.0 ± 0.5) treatments. We showed that during in vitro capacitation of cattle: (a) NO influences sperm motility and vigour; (b) NO is associated with cGMP synthesis through two independent pathways and (c) the cGMP/PKG1 pathway has a partial role in sperm capacitation and does not involve the L‐arg/NO.     

鸡贮精腺上皮细胞的分离及原代培养

试验旨在探讨稳定可靠的贮精腺上皮细胞分离及原代培养方法,为研究鸡贮精机理提供细胞模型。以鸡输卵管的子宫阴道交接部组织样为材料,采用酶消化法和组织块培养法分离培养母鸡贮精腺上皮细胞,观察母鸡贮精腺上皮细胞的培养情况,比较不同细胞培养方法获得贮精腺上皮细胞的生长情况。结果表明,用胶原酶或胰酶单独消化母鸡子宫阴道交接部组织,经100目过滤后获得的贮精腺上皮细胞24 h后可贴壁,但48～72 h后细胞死亡;用胶原酶Ⅺ(0.01 g/mL)与胰酶(0.25%)先后消化母鸡子宫阴道交接部组织后再经100目过滤获得的贮精腺上皮细胞贴壁性良好,24～48 h细胞出现明显增殖,72 h后细胞增殖速度减慢,开始死亡;用组织块培养法7 d可获得鸡贮精腺上皮原代细胞,该细胞可传2～3代;用组织块培养法获得的细胞进行免疫组化试验,发现细胞表达贮精腺差异表达基因编码的NXPH1蛋白,该蛋白在培养细胞内的表达符合其分泌蛋白特性,表明组织块培养法所获细胞可用于后续研究。综上,用组织块培养法获得的鸡贮精腺上皮细胞可为研究母鸡贮精腺机制提供细胞模型。     

Birth of puppies of predetermined sex after artificial insemination with a low number of sex‐sorted,frozen–thawed spermatozoa in field conditions

The aim of this study was to evaluate fertility and sex ratios after artificial insemination in dogs under field conditions. Semen was cryopreserved as unsorted (control) or was separated into X‐ and Y‐chromosome‐bearing sperm using a cell sorter. Sixty female dogs were inseminated with frozen–thawed spermatozoa of 100 × 10⁶ unsorted (a dose in practice) and 4 × 10⁶ sorted (X and Y group, respectively). A total of 20 dogs became pregnant and 126 puppies were born from the three groups. The percentage of parturition was similar for the X (5/20; 25.0%) and Y (4/20; 20.0%) group (P > 0.05), but lower than controls (11/20; 55.0%) (P < 0.05). Ultimately 28 out of the 32 puppies produced from X group were female (87.5%) and 19/22 (86.4%) puppies of Y group were male. In contrast, sex ratio (51.4% to 48.6%) in the control was significantly different from the X, Y group (P < 0.05). However, male and female puppies in the control had similar birth weights and weaning weights to those from the X and Y groups. This preliminary information indicated that normal puppies of predicted sex can be produced with low numbers of sorted cryopreserved dog spermatozoa at a farm level, making sperm‐sexing technology potentially applicable for elite breeding units.