不同品系小鼠精子冷冻及冻融精子体外受精的研究

采用小鼠精子冷冻、冻融精子体外受精的方法,进行小鼠保种、引种、生物净化及品系标准化的可行性研究,并初步探究小鼠精子冷冻的影响因素。实验选用同一年龄5种不同品系小鼠分别进行精子冷冻、复苏,并比较各品系冷冻效果及冻融精子的体外受精(IVF)率。结果显示,冷冻后各品系小鼠精子复苏存活率约在15%～50%之间;冻融小鼠精子体外受精结果随遗传背景的不同差异显著,受精率分别为:KM小鼠68.4%、ICR小鼠30.3%、DBA/2J小鼠47.2%、C57BL/6J小鼠6.8%、BALB/cA小鼠25%。冻融精子IVF得到的胚胎可以耐受体外培养。     

小鼠精原细胞体外培养的研究

采用不同的酶分步消化,分离培养小鼠的支持细胞和精原细胞,并根据它们的不同特性采用不同的染色方法进行鉴定.利用支持细胞对精原细胞的支持和营养作用,使支持细胞和精原细胞共培养,对精原细胞在体外分化时的条件要求作了研究.结果:获得了能进行分化的精原细胞,而且已经分化到个体变小,细胞核偏向一侧的细胞.结论:通过体外培养精原细胞获得精子细胞是可行的.     

Fertilization of rabbit ova in vitro by sperm with adsorbed Sendai virus

Fertilization occurs when rabbit ova are cultured in vitro with epididymal sperm to which Sendai virus is adsorbed. These sperm do not require capacitation in vivo in order to fertilize. Evidence for fertilization is penetration of sperm, the appearance of two polar bodies and pronuclei, and cleavage through the eight-blastomere stage. The viruses attach almost exclusively to the sperm acrosome, with resultant head-to-head agglutination of the sperm.     

Circadian system controlling release of sperm in the insect testes

Release of mature sperm from the testis into seminal ducts of the gypsy moth exhibits a circadian rhythm. The rhythm of sperm release was shown to persist in vitro, in isolated complexes of testis and seminal ducts cultured in light-dark cycles or in constant darkness. The phase of the rhythm was also reset in vitro by exposure to shifted light-dark cycles. Therefore, the testis-seminal ducts complex from the gypsy moth is photosensitive and contains a circadian pacemaker, which controls the rhythm of sperm movement. This finding extends the range of structures in multicellular organisms that are known to contain circadian oscillators and provides a new model system in which circadian mechanisms may be studied.     

不同冷冻-解冻精子浓度对猪卵母细胞体外受精的影响

[目的]探讨体外受精时不同精子浓度对精子穿入成熟卵母细胞及后续发育的影响。[方法]以冷冻-解冻精子为材料,以1×106、1×107个/ml 2种精子浓度进行体外受精,研究不同冷冻-解冻精子浓度对猪卵母细胞体外受精的影响。[结果]当精子浓度为1×106、1×107个/ml时,精子穿入率分别为83.18%、88.23%,单精入卵率分别为40.55%、9.02%,多精入卵率分别为59.45%、90.98%,平均每个卵母细胞中的精子数分别为2.03、3.26个,雄原核形成率分别为84.10%、86.40%,单精入卵率、多精入卵率有显著差异(P<0.05),其他指标无显著差异(P>0.05)。[结论]该研究为应用冷冻-解冻精子建立猪卵母细胞的体外生产系统提供了依据。     

不同因素对牛卵母细胞胞质内精子注射效果的影响

采用胞质内精子注射法（ICSI）构建牛显微受精胚胎,探讨卵母细胞成熟培养时间、精子状态及精子操作液中聚乙烯吡咯烷酮（PVP）浓度对牛卵母细胞胞质内显微受精（ICSI）卵体外发育的影响。结果显示：成熟培养20 h、24 h和28 h的卵母细胞用于ICSI时,ICSI卵的形态完整率分别为94.6%（175/185）、97.2%（173/178）和96.4%（189/196）（P〉0.05）;成熟培养24 h和28 h的卵母细胞,其ICSI卵的卵裂率（73.4%和70.9%）和囊胚发育率（31.8%和29.6%）显著高于成熟培养20 h的卵母细胞（61.1%和20.6%）（P〈0.05）。对成熟培养24 h的卵母细胞注射获能精子时,ICSI卵的卵裂率和囊胚发育率（72.7%和31.4%）高于未获能精子组（69.4%和29.3%）和不运动精子组（71.5%和30.4%）,但无统计学差异（P〉0.05）。采用含5%,10%和15%PVP的精子操作液处理获能精子后对成熟培养24 h的卵母细胞进行ICSI操作,PVP浓度为5%和10%时,ICSI卵的卵裂率（73.2%和66.9%）和囊胚发育率（32.7%和29.7%）差异不显著（P〉0.05）,但二者均显著高于15%PVP浓度组的卵裂率和囊胚发育率（51.0%和16.3%）（P〈0.01）。结论认为,选用获能精子,并使用含5%PVP的精子操作液进行处理,对成熟培养24 h的牛卵母细胞进行ICSI操作,有利于ICSI卵的体外发育。     

睾丸支持细胞对体外精子活力的影响

通过建立小鼠睾丸支持细胞与精子共培养的方法,达到体外提高精子活力的目的.利用睾丸组织切片技术,观察并分析支持细胞对生精细胞的作用.从6~7日龄小鼠睾丸中分离睾丸支持细胞,体外培养两天后,形成细胞单层作为滋养层.取小鼠附睾尾内精子,转移到以支持细胞为滋养层的培养液中培养,同时设立对照组.通过精子分析仪观察各组精子的活力,结果显示,以支持细胞为滋养层的精子前向运动百分比为56.1±5.81%,显著高于对照组40.7±2.96% (P<0.05).利用激光共聚焦显微镜观察精子结构,结果显示两组精子形态结构差异不显著,表明支持细胞能够提高体外精子的活力,但不改变精子的形态结构.     

Transglutaminase-mediated modifications of the rat sperm surface in vitro

Two transglutaminase-mediated modifications of the rat epididymal spermatozoon surface were demonstrated in vitro. Transglutaminase was effective in promoting the binding of spermidine to the sperm. Moreover, the enzyme, by reacting with one of the major proteins secreted by the rat seminal vesicle epithelium, produced a modified form of the protein with a higher molecular weight and the capability of binding to the sperm cells. A specific physiological role for the enzyme, bringing about modifications of the rat sperm surface in the seminal fluid environment, is suggested.     

不同获能液对猪精子获能效果的初步观察

为了探讨不同获能液对猪精子获能效果的影响,以新鲜猪精液为材料,进行mTBM＋咖啡因、mTBM＋咖啡因＋BSA,mTBM＋肝素三种获能液获能效果的比较。利用金霉素荧光染色法对其获能效果进行检验。结果表明,mTBM＋肝素组诱发B型（获能）精子率极显著高于mTBM＋咖啡因组（P〈0.01）,显著高于mTBM＋咖啡因＋BSA组（P〈0.05）;三种获能液诱发的AR型（顶体反应）精子率差异不显著（P〉0.05）。说明,添加肝素更有助于提高猪精子获能效果。     

Boer山羊精子体外获能前后超微结构的研究

用S培养体系对Boer山羊精子进行体外获能试验，借助电子显微技术观察其获能前后超微结构的变化，结果表明，Boer山羊精子的超微结构与其它哺乳动物的类似，分头、颈、尾3部分。头部主要由细胞核构成，含有遗传信息；颈部脆弱，尾部易从此脱落；尾部是精子运动的器官，由中段、主段和末段3部分组成，精子获能后，顶体质膜膨胀、断裂和丢失；顶体外膜囊泡化，进而顶体内膜裸露。     

一个重要的精子受体—猪透明带糖蛋白β-D-半乳糖残基

 【目的】证明末端半乳糖残基在猪透明带糖蛋白中的分布以及它在精子结合和侵入中的作用。【方法】体外成熟的猪卵母细胞去除周围卵丘细胞，用异硫氰酸荧光素(fluorescein isothiocyanate，FITC) 络合西非单叶豆凝集素(Bandeiraea simplicifolia lectin-I，BS-I) 或蓖麻凝集素(Ricinus communis agglutinin，RCA-I)处理30min，分别观察末端-D-半乳糖残基或-D-半乳糖残基在透明带中的定位分布。荧光显微镜观察显示，BS-I和RCA-I都标记于整个透明带上且比较集中于透明带的外层，其中BS-I显示微弱荧光反应，而RCA-I呈强荧光反应且在透明带的外层反应尤为强烈。植物凝集素处理的卵母细胞用于体外受精，分别在受精2h或12h观察精-卵结合和精子入卵情况。【结果】RCA-I处理组卵母细胞平均精子结合数显著低于对照组（18 vs 95），而精子入卵则完全被阻断；可是BS-I处理组的卵母细胞的精子结合数少量被抑制（59 vs 95），而且精子入卵率与对照组无显著差异。【结论】猪透明带末端-D-半乳糖残基作为精子受体的重要组成成分，参与精-卵结合和受精。     

Monoclonal antibody to a human germ cell membrane glycoprotein that inhibits fertilization

A monoclonal antibody to an antigen in the human germ cell membrane did not agglutinate or immobilize sperm but inhibited binding and penetration of zona-free hamster ova by human sperm and blocked murine fertilization in vitro. The antibody, of the 2a subclass of immunoglobulin G, was germ cell-specific but not species-specific. It recognized a single antigen of 23 kilodaltons that has been isolated from human germ cells. This fertilization antigen, located on the postacrosome , midpiece, and tail of human sperm, is a glycoprotein of testicular origin associated with some types of human involuntary immunoinfertility .     

RNA Interference-Mediated Downregulation of sAC Gene Inhibits Sperm Hyperactivation in Male Rats （Rattus norvegicus）

Hyperactivation is one of the most critical parts for fertilization, cAMP generated by soluble adenylyl cyclase （sAC） is necessary to activate sperm and is a prerequisite for sperm hyperactivation. The aim of this study is to investigate the function of sAC in hyperactivation in male rats. Four siRNAs of sAC gene were designed and separately transformed into rat sperm using electrotransformation method. Cultured for 12 and 24 h, physiological and biochemical indexes of these sperm were analyzed, and the expressions of some hyperactivation-related genes were detected using real-time PCR. We demonstrated 26.3-30.8% and 49.1-50.5% reduction in sAC at the protein by Western blot and mRNA levels by real-time PCR, respectively. The results showed that two siRNAs, Actb-717 and Actb-4205, were the best RNAi sites for silencing sAC. The VCL （curvilinear velocity） and ALH （amplitude of lateral head displacement） of RNA interference （RNAi）-transfected sperm were reduced, cAMP and protein phosphorylation in RNAi transfected sperm were also decreased. The hyperactivation-related genes, such as CatSper2, LDHC and PKA, were downregulated in the sperm, which sAC was knockdown. These findings demonstrated that sAC might play a critical role in cAMP signaling in the rat sperm hyperactivation, and downregulated sAC gene might prevent the expression of these hyperactivation-ralated genes resulting in sperm dysfunction. These findings suggest that these hyperactivation-ralated genes and sAC are functionally related in sperm hyperactivation and sAC falls into an expanding group of sperm proteins that appear to be promising targets for the development of male contraceptives.     

Generation and in vitro differentiation of a spermatogonial cell line

Spermatogenesis is the process by which spermatogonial stem cells divide and differentiate to produce sperm. In vitro sperm production has been difficult to achieve because of the lack of a culture system to maintain viable spermatogonia for long periods of time. Here we report the in vitro generation of spermatocytes and spermatids from telomerase-immortalized mouse type A spermatogonial cells in the presence of stem cell factor. This differentiation can occur in the absence of supportive cells. The immortalized spermatogonial cell line may serve as a powerful tool in elucidating the molecular mechanisms of spermatogenesis. Furthermore, through genomic modification and transplantation techniques, this male germ cell line may be used to generate transgenic mice and to develop germ cell gene therapy.     

奶山羊精子体外获能与体外受精

供体用FSH+LH超排,卵母细胞从注LH后约30h回收。将获能精子与10枚卵母细胞在受精液(mDM+输卵管上皮细胞)和培养液(mDM+30％供体羊血清)中连续培养(38℃)24h后,有6枚卵受精,抛出第二极体。将6枚受精卵移植到3只受体中,有1只妊娠,产出1只试管母山羊。初步表明,本试验的奶山羊精子体外获能与体外受精程序是简便而有效的。     

Human CHN1 mutations hyperactivate alpha2-chimaerin and cause Duane's retraction syndrome

Duane's retraction syndrome (DRS) is a complex congenital eye movement disorder caused by aberrant innervation of the extraocular muscles by axons of brainstem motor neurons. Studying families with a variant form of the disorder (DURS2-DRS), we have identified causative heterozygous missense mutations in CHN1, a gene on chromosome 2q31 that encodes alpha2-chimaerin, a Rac guanosine triphosphatase-activating protein (RacGAP) signaling protein previously implicated in the pathfinding of corticospinal axons in mice. We found that these are gain-of-function mutations that increase alpha2-chimaerin RacGAP activity in vitro. Several of the mutations appeared to enhance alpha2-chimaerin translocation to the cell membrane or enhance its ability to self-associate. Expression of mutant alpha2-chimaerin constructs in chick embryos resulted in failure of oculomotor axons to innervate their target extraocular muscles. We conclude that alpha2-chimaerin has a critical developmental function in ocular motor axon pathfinding.     

水牛受精卵胞质内注射转基因的初步研究

[目的]旨在探讨通过向水牛受精卵胞质内注射外源DNA实现转基因的可行性。[方法]水牛卵母细胞体外成熟20～22 h后随机分为2组:①在体外受精7～10 h或18～20 h后向卵胞质内注入约7.5 pl 50μg/ml含线性EGFP片段的DNA溶液;②分别注入单个精子与约7.5 pl 50μg/ml含线性EGFP片段的DNA混合物,观察外源基因在胚胎发育过程中的表达情况。[结果]受精卵胞质内注射的早期胚胎基因表达率、囊胚基因表达率与ICSI-Tr差异不显著(P0.05),且IVF 7～10 h时注射的分裂率、早期胚胎基因表达率均显著高于18～20 h(P0.05)。[结论]水牛IVF受精卵胞质内注射外源基因能获得转基因胚胎,且IVF后7～10 h注射的效果优于IVF后18～20 h注射。     

黑尾近红鲌精子活力研究

在水温23℃的条件下,对黑尾近红鲌(Ancherythroculter nigrocauda Yih et Wu)精子在不同浓度氯化钠溶液、不同水体以及不同离体时间情况下的活力进行了研究。结果表明,1在0~1.2%的氯化钠溶液中,氯化钠浓度小于0.5%时,随着浓度的增加,黑尾近红鲌精子快速运动时间随之延长;在0.5%氯化钠溶液中,精子快速运动时间最长(52.00±3.46 s),与其他组别差异显著(P0.05);当浓度大于0.9%时,精子未激活。2精子在蒸馏水中的快速运动时间(36.33±1.15 s)显著(P0.05)高于在自来水(32.00±1.00 s)和池塘水(27.67±1.52 s)中。3采用蒸馏水激活离体精子1~15 h,随着时间的延长,存活精子快速运动时间无显著差异(P0.05),而精子成活率显著降低(P0.05)。     

Inhibition of sperm-egg interaction by specific antibody

Treatment of hamster ova, in vitro, with ovary specific antibody interferes with sperm-egg interaction by inhibiting sperm attachment to the zona pellucida and subsequent zona penetration. This inhibition results from precipitation of the zona by the specific antibody.     

Cell biology. A universal bicarbonate sensor

The life-span of sperm may be short but it is certainly busy. The three principal molecular events that prepare sperm for fertilization are all controlled by the intracellular nucleotide adenosine 3',5'-monophosphate (cAMP). One of these, capacitation, is also regulated by bicarbonate ions. The elusive connection between cAMP and bicarbonate ions now appears to be solved as Kaupp and Weyand explain in their Perspective. Bicarbonate ions enter sperm through the anion transporter in the sperm plasma membrane and activate the soluble form of adenylyl cyclase, the enzyme that synthesizes cAMP (Chen et al.)