异源精子诱导栉孔扇贝雌核发育早期胚胎发育及其倍性分析

采用紫外线遗传灭活的长牡蛎（Crassostrea gigas）精子作激活源,经6-DMAP加倍诱导栉孔扇贝（Chlamys farreri）第二极体抑制型雌核发育二倍体;运用荧光显微技术,对雌核发育二倍体卵子早期胚胎发育过程中的核相变化进行观察。结果显示,紫外线处理过的精子入卵后发生一次轻微膨胀,形成雄性原核,但不形成染色体,而是浓缩为致密的染色质小体（DCB）,DCB或滞留于两卵裂球的分裂沟上或进入其一的细胞质中,不与雌原核融合;雌核发育胚胎的发育速度明显滞缓,经6-DMAP处理后胚胎发育速度加快,发育同步化;在雌核发育二倍体诱导过程中,还观察到杂交体、异源三倍体、雌核发育单倍体、雌核发育四倍体。结果为异源精子诱导栉孔扇贝雌核发育提供了细胞学证据。     

Appraisal and standardization of curvilinear velocity (VCL) cut‐off values for CASA analysis of Japanese quail (Coturnix japonica) sperm

One of the basic steps in objective analysis of sperm motility is the subdivision of a motile sperm population into slow, medium and rapid categories based on their velocity. However, for CASA analysis of quail sperm, the velocity values for categorization of slow, medium and rapid sperm have not yet been standardized. To identify the cut‐off values of “velocity curvilinear” (VCL) for quail sperm categorization, we captured and analysed 22,300 tracks of quail sperm using SCA^®‐CASA. The median and mean VCL values were 85 and 97 μm/s. To define the VCL cut‐off values, we used two methods. In the first, we identified the upper (rapid sperm) and lower (slow sperm) cut‐off values using: (i) median VCL ± 25% or ± 50% or ± 75% of median VCL value; (ii) first and third quartile values of VCL data (i.e. 25% cut‐off setting); and (iii) 33% and 66% of VCL data. Among these settings, sperm categories and their corresponding motility characteristics recorded using the “25%” setting (i.e. slow ≤36 ≤ medium ≤154 ≤ rapid) were found the most realistic and coherent with male ranking by fertility. In the second method, we calculated heteroscedasticity in the total VCL data using PCA and the two‐step clustering method. With this approach, the mean of the high and low clusters was 165 and 51 μm/s, respectively. Together, the mean from two methods suggested that, for SCA^®‐CASA categorization of quail sperm, sperm should be classed as “rapid” at VCL ≥160 μm/s and “slow” at VCL ≤45 μm/s.     

Motility of sea urchin Paracentrotus lividus spermatozoa in the post‐activation phase

The characterization of sperm motility patterns, particularly post‐activation changes, is the first step in setting up species‐specific protocols involving gamete management and embryo production, for both aquaculture and laboratory research purposes. This study is aimed at the characterization of the sperm motility pattern of the purple sea urchin Paracentrotus lividus. Semen samples were individually diluted in artificial sea water for sperm motility activation. They were then incubated at 18°C for up to 24 hr. Motility was evaluated on dilution, and 1 hr, 3 hr and 24 hr after activation, by computerized analyser. The semen fertilization capacity was also evaluated. Under our experimental conditions (dilution 1:1,000 in artificial sea water plus 0.05% BSA, 18°C, in the dark), P. lividus semen remained viable for up to 24 hr, as the total motile sperm and the fertilization percentages did not change significantly during the incubation time. In contrast, the mean curvilinear velocity and the subpopulation of rapid sperm (those having a curvilinear velocity > 100 µm/s) slightly but significantly decreased after 3 hr, thereafter remaining unchanged for up to 24 hr after activation. In conclusion, our results show that diluted P. lividus semen can be used for a longer period than that of most fish species, with no need for motility inhibition procedures, supporting its wider use in laboratory research. In addition, the development of artificial fertilization protocols for aquaculture production is simplified by long‐lasting sperm motility.     

棉铃虫精子的超微结构及对其辐照后变化的初步观察

利用透射电子显微镜技术,研究了棉铃虫（ＨｅｌｉｃｏｖｒｐａａｒｍｉｇｅｒａＨｕｅｂｎｅｒ）真核精子和无核精子的超微结构,比较了在经γ射线照射的棉铃虫精子与未照射虫精子结构的差异,结果表明,棉铃虫真核精子和无核精子的顶体结构有较大差异。真核精子有核,呈半月形,鞭毛线粒体衍生物与微管系统紧密结合,从头到尾在质膜上都覆盖许多特定结构的外长物;无核精子没有核,顶体和鞭毛通微管连接,鞭毛缄粒体衍生物与微管系     

Sperm production and quality in European eel (Anguilla anguilla) in relation to hormonal treatment

Aquaculture production relies on controlled management of gametogenesis, especially in species where assisted reproduction is needed for obtaining gametes in captivity. The present study used human chorionic gonadotropin (hCG) treatments to induce and sustain spermatogenesis in European eel (Anguilla anguilla). The aim was to evaluate effects of strip-spawning timing (12 vs. 24 hr) after weekly administration of hCG and the necessity of a primer dose (in addition to weekly hormonal treatment) prior to strip spawning (primer vs. no-primer) on sperm quality parameters. Sperm parameters included milt production (weight), density and sperm kinematics at Week 9, 11 and 13 after onset of treatment. Spermiation commenced in 11.5% of males in Week 5 and by Week 9, and all males produced milt. Male weight, milt production, sperm density and spermatocrit did not differ among hormonal treatments during the experimental period. Overall, male weight decreased from 106.3 to 93.0 g, milt weight increased from 3.5 to 5.4 g, sperm density counts decreased from 11.7 × 10⁹ to 10.5 × 10⁹ cells/ml, and spermatocrit decreased from 46.5% to 40.5%. Furthermore, spermatocrit was positively related to haemocytometer counts (R² = .86, p < .001), providing a reliable indicator of sperm density. Differences in sperm kinematics were observed depending on strip-spawning timing after hormonal injection (12 vs. 24 hr) but with no consistent pattern. These sperm quality parameters also did not consistently differ between the no-primer and primer treatments. Considering that each male may be stripped 4–5 times over the 2–3 months spawning season, omitting the primer would reduce animal handling, material costs and labour intensity, while sustaining high-quality sperm production.     

水牛精子在胞质内注射后的早期形态变化

应用胞质内精子注射 (intracytoplasmic sperm injection,ICSI)技术探讨了水牛卵母细胞胞质与注射精子间的相互作用以及 ICSI精子的形态变化。ICSI后 13h,5 9.4 %的精子头部已膨大 ,且有 18.8%的精子进入解聚状态。雌雄原核发育不同步 ,雄原核在 ICSI后 16 h和 19h的形成率分别为 3.2 %和 4 0 .0 % ;雌原核在 ICSI后 13h已达到 71.9% ,16 h时提高到 90 .3%。经离子霉素与 6 - DMAP联合激活 ,ICSI后 19h产生的胚胎核型以 2 PN PB1 为主 ,其雌原核形成 (激活 )率为 91.3% ,雄原核形成率为 4 0 .0 % ,5 1.3%为孤雌胚。用 5 mm ol/ L 的 DTT预处理精子 1h,可提高精子的解聚率 (30 .9%比 12 .7% ,P<0 .0 5 ) ,但对雄原核的形成率无显著影响 (33.3%比 32 .7% ,P>0 .0 5 )。结果表明 ,水牛精子 ICSI后的雄原核形成时间晚于卵子雌原核形成时间 ,且其比率低于卵子 ,DTT处理精子能提高其解聚率 ,但对雄原核形成无影响     

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.     

Evidence of LAT1 expression in canine caput epididymis

L-type amino acid transporter 1 (LAT1), the first isotype of amino acid transport system L, transports aromatic and branched amino acids pivotal for fundamental cellular activities such cellular growth and proliferation. LAT1 expression was high only in the brain in contrast to its limited distribution and low level of expression in normal tissues. We found potent LAT1 expression in canine caput epididymis by quantitative RT-PCR and Western blotting analysis. Immnuno-histochemical examination revealed observable LAT1 in microvillous epithelial cells.     

牛精子体外获能前后元素变化的研究

 本文应用x射线能谱技术，对获能前后精子质膜表面（头部、中段）和内部（顶体内、中段线粒体内）的元素进行了分析测试。结果表明，牛精子用肝素+咖啡因体外获能后，头部、中段质膜表面的Na、Al、S、K含量升高，Cl、Ca含量降低；顶体内Na、Mg、Si、P和Fe含量降低，而Ca、K、Cl和S含量升高；精子中段线粒体内Na、P、Si、Cl和Ca升高，而Mg、S、K和Fe却降低。获能过程中精子顶体内Na/Ca、Na/K、Cl/Ca、Cl/K和Na/Cl的浓度比分别为42.13、12.43、15.36、45.31和2.74；而摩尔比又分别为73.42、21.13、17.37、4.99和4.23。本文并对可能的获能机理进行了讨论。     

不同冻融处理对猪精子顶体膜蛋白表达的影响

为探究不同冷冻解冻方法对猪精子损伤的影响,采用2种常用的冷冻解冻方法处理猪精子,分别对其顶体膜蛋白进行分离,进行SDS-PAGE电泳和总灰度值的检测和分析。结果表明,采用一次稀释法进行猪精液的冷冻保存较二次稀释法解冻后精子活率高,精子顶体膜蛋白组分中除125 ku和90 ku处蛋白表达水平低于二次稀释法外,120 ku、48 ku、36 ku均高于二次稀释法。可见,猪精子在受到更深层次(二次稀释法对精子冷冻解冻损伤大)的冷冻损伤时,伴随着顶体膜蛋白125 ku和90 ku表达水平的升高以及120、48及36 ku表达水平的降低。