人精液中生精细胞染色体两种制备方法

人精液中生精细胞染色体两种制备方法庞铁石人减数分裂期（生精细胞和精子）染色体分析是遗传学、组织胚胎学、细胞生物学和生殖生物学研究的重要手段。以往只能采用精子－地鼠卵体外受精法检查成熟精子单倍染色体。相比之下，生精细胞（精原细胞、初级／次级精母细胞、精...     

直接低渗法制备男性不育症精液生精细胞染色体观察

目的：研究男性不育症生殖细胞减数分裂过程、生精细胞染色体畸变与不育的关系。方法：选择不育门诊中１９例男性原发不肓患者精液和４例禁欲期正常男性志愿者精液，应用直接低渗法，获得各级生精细胞（精原细胞、初级／次级精母细胞、精细胞）分裂相。结果：不同样本精液中各级生精细胞分裂相分布有极显著性差异（Ｐ〈０．００５）；弱精症患者精液标本中主要为ＭＩ单价体、染色单体畸变，少精症和无精症主要为联会消失、减数分裂阻断。结论：直接低渗法能为诊断男性不育提供有价值的细胞遗传学信息。生精细胞染色体畸变、减数分裂过程异常是导致男性不肓的原因之一。     

生精细胞的凋亡与调控

生精细胞凋亡是近年来生殖生物学的研究热点之一 ,动物的不同发育阶段 ,年龄差异 ,激素水平尤其FSH是影响生精细胞凋亡的重要因素。本文重点对生精细胞凋亡的影响因素及其基因调控机制进行综述。     

凋亡生精细胞的观察

用瑞－姬法观察凋亡生精细胞的各种形态，即凋亡的初级精母细胞、次级精母细胞、精子细胞，并观察了凋亡的前列腺上皮细胞的形态，就生精细胞凋亡原因进行了分析。     

生精细胞凋亡及其调控

在细胞生物学领域,细胞凋亡的实现调控着细胞死亡和增殖的平衡,故具有重要的生物学意义。生精细胞的凋亡在生精过程中具有极其重要的作用。近年来,睾丸内细胞凋亡的研究在国内外已成为热点之一。本文将就生精细胞凋亡的的阶段性、生理意义、形态特点、激素调节、基因调控等诸多方面的研究概况作一综述。     

实验性隐睾诱导小鼠生精细胞凋亡的研究

目的 研究手术隐睾所致成年小鼠生殖细胞凋亡及相关调节因子ｂｃｌ－２、ｂａｘ蛋白在曲细精管中的定位、变化。方法 以末端脱氧核糖核酸转移酶介导的ｄＵＴＰ缺口末端标记法（ＴＵＮＥＬ）检测凋亡的生殖细胞,生物素－抗生物素蛋白ＤＣＳ体系间接免疫荧光法检测Ｂｃｌ－２、Ｂａｘ蛋白在曲细精管中的定位、分布。结果 隐睾术后３ｄ,手术侧睾丸重量及细胞凋亡数量与对侧地无明显区别。而６￣１５ｄ,睾丸重量明显减轻,凋亡细胞     

小鼠染色体制备方法初探

目的改进小鼠染色体制备过程中的关键环节,提高分裂中期染色体制备效果。方法严格选材,改进小鼠实验前用药剂量及时间,控制滴片高度,把握染色时间。结果染色体颜色呈鲜艳的玫瑰红色,形状呈均匀的圆盘状分布。结论掌握染色体制备的原理是改进实验方法的基础,严格规范操作方法是关键。     

小鼠染色体制备方法初探

目的改进小鼠染色体制备过程中的关键环节,提高分裂中期染色体制备效果。方法严格选材,改进小鼠实验前用药剂量及时间,控制滴片高度,把握染色时间。结果染色体颜色呈鲜艳的玫瑰红色,形状呈均匀的圆盘状分布。结论掌握染色体制备的原理是改进实验方法的基础,严格规范操作方法是关键。     

辐射致雄性小鼠不育症模型的建立及生精细胞损伤的初步分析

目的：构建辐射致雄性小鼠不育症模型,并对模型小鼠的生精细胞损伤进行初步分析。方法：不育症模型构建：65只雄性C57BL/6小鼠随机分为7组,正常对照组(A组)及6组60Co γ照射组,后者包括B组(12 Gy)、C组(6+6 Gy)、D组(10 Gy)、E组(6+4 Gy)、F组(8 Gy)、G组(4+4 Gy)。观察分析小鼠死亡情况(24w)及30、50、70 d受孕率。生精细胞损伤分析：72只雄鼠随机分为4组：正常对照组、6+4 Gy组、4+4 Gy组、6 Gy组。30、50、70 d观察睾丸组织切片(HE染色)并评估其生精功能( Johnsen评分);对PLZF阳性标记的精原干细胞(免疫组化法)计数。结果：A、E、F、G组生存时间较长,B、C组生存时间较短。30、50、70 d,D、E组受孕率均为0%。各照射组Johnsen评分均低于对照组(P<0.05);6+4 Gy组的PLZF阳性细胞计数呈逐渐上升趋势,但均明显低于对照组(P<0.05)。结论：6+4 Gy的60Co-γ射线照射7周龄的C57BL/6雄性小鼠,可以构建不育症模型。模型小鼠的PLZF标记的精原干细胞计数虽然低于对照组,但并非完全消失,提示不育症的原因并非精原干细胞的完全消亡。     

Stability of transferred human chromosome fragments in cultured cells and in mice

Chromosome fragments represent feasible gene delivery vectors with the use of microcell-mediated chromosome transfer. To test a prerequisite for a gene delivery vector, we examined the stability of human chromosome fragments (hCFs) in cultured cells and in trans-chromosomic (Tc) mice. Fragments of human chromosomes 2 (hCF(2-W23)), 11 (hCF-11) and 14 (hCF(SC20)) tagged with neo were introduced into the TT2F mouse ES cells, and retention of the hCFs was examined by FISH during long-term culture without selection. In contrast to the gradual loss of hCF(2-W23) and hCF-11, hCF(SC20) remained stable over 70 population doublings in the ES cells. The hCF(SC20) was also stable in cultured human tumor cells and chicken DT40 cells. We have previously generated chimeric mice using the ES cells harboring the hCF(2-W23) or hCF(SC20), followed by production of Tc mice. Although both the hCF(2-W23) and hCF(SC20) persisted in cells of Tc mice as an additional chromosome and were transmitted to offspring, the hCF(SC20) was more stable than the hCF(2-W23) in F1 and F2 mice. The present study shows that the stability of hCFs in Tc mice differs with tissue types and with genetic background used for successive breedings. Thus, the hCF(SC20), which was relatively stable in both mouse and human cells, may be a promising candidate for development as a gene delivery vector. This revised version was published online in July 2006 with corrections to the Cover Date.     

Variation of mouse oocyte sensitivity to griseofulvin-induced aneuploidy and meiotic delay during the first meiotic division

The effects of varying the time of chemical treatment on the induction of areuploidy and meiotic delay in metaphase II (Mll) oocytes were studied by administering 1,500 mg/kg griseofulvin (GF) at 0, 2, 4, 6, or 8 hr after on injection of human chorionic gonadotrophin (HCG). The results show that the oocytes have a different sensitivity to GF-induced aneuploidy and meiotic delay during the course of meiotic maturation. Although not restricted to a particular period of meiotic maturation, the frequency of aneuploidy was highest (P < 0.05) when GF was given at 2, 4, or 6 hr after HCG. The maximum frequency of hyperploidy (42.4%) occurred at the 4-hr treatment time. Also, GF treatment resulted in the induction of meiotic delay as demonstrated by ovulated metaphase I (Ml) and polyploid Mll oocytes. The meiotic delay data depict a period of relative resistance between two periods of sensitivity in that the percentages of ovulated Ml oocytes were 53.3, 21.3, 3.5, 6.7, and 25.7 when GF was given at 0, 2, 4, 6, and 8 hr after HCG, respectively. Also, at these treatment times the percentages of polyploid oocytes were 0.6, 1.7, 7.7, 20.1, and 15.4, respectively. Therefore, the oocytes seem to be more sensitive to GF-induced meiotic delay during the periods preceding and following meiotic spindle assembly. In conclusion, the results demonstrate that the time of chemical treatment influences the frequency of aneuploidy and the degree of meiotic delay. Also, the results emphasize that to thoroughly characterize the aneugenic potential of a specific chemical several treatment times may be needed. © 1994 Wiley-Liss, Inc.     

Intrauterine insemination and comparison of two methods of sperm preparation

In a study of intrauterine inseminations (IUI) after clomiphene stimulation, a randomized comparison was made between a new method of sperm preparation, self migration in sodium hyaluronate (SH), and a traditional method, centrifugation and swim up (CS). After two IUI cycles with either SH or CS, the sperm preparation method was swapped and the patients received another two IUI cycles. Interjacent cycles of natural intercourse after clomiphene treatment served as the control. The SH method resulted in a significantly higher percentage recovery of progressive motile spermatozoa than the CS method, 17.7% versus 8.6% (P less than 0.01). The sperm samples were prepared by SH in 68 cycles and by CS in 57 cycles, resulting in six and five pregnancies, respectively. Pregnancies were obtained in 11 of 125 IUI cycles (8.8%) and in 3 of 124 control cycles (2.4%) (P less than 0.05). The pregnancy rate following IUI was highest in the patients with cervical factor (35%) and asthenozoospermia (23%), while none became pregnant in the group with oligozoospermia. In the unexplained infertility group, no difference between the pregnancy rates in IUI cycles and control cycles was seen. SH is a simple and rapid method of sperm preparation and it appears to give a high recovery of motile spermatozoa and a number of pregnancies which is comparable to that of CS. Treatment with IUI in cycles with a simple stimulation protocol seems to be valuable in cases involving either a cervical factor or asthenozoospermia.     

连续量化外周血淋巴细胞染色体的制备方法

目的 快速制备外周血淋巴细胞染色体,提高分析效率,为染色体制备的规范化标准化提供理论依据.方法 连续量化法在传统制备方法基础上去掉三次室温放置固定时间,滴片后随之烤片,提高秋水仙素终浓度(0.008 μg/mL)等方面进行了优化,同时与传统制备法进行对比.结果 278例染色体标本:传统组400~600条分裂相占63%,连续量化收获组占65%(P=0.065);传统组染色体分散良好率65%,连续量化收获组良好率66%(P=0.088);两组比较无显著差异,表明两种方法无差别.结论 连续量化外周血淋巴细胞染色体的制备方法重复性和一致性好,缩短了染色体制备过程,提高了染色体分析的效率.     

杂色曲霉素对小鼠穹窿下器室管膜细胞超微结构的影响

目的：观察杂色曲霉素（ST）对小鼠穹隆下器（SFO）室管膜细胞超微结构的影响。方法：BALB／c小鼠,单次ST（3000μg/kg）灌胃,分别于灌胃后1、2、4、8、16h处死动物,用扫描电镜观察SFO室管膜的结构变化。结果：ST灌胃后1h部分SFO室管膜表面微绒毛结构消失、细胞破损,ST灌胃后2h室管膜出现凹凸不平、损伤加重。ST灌胃后8h室管膜凹凸不平和破损最为明显,ST灌胃后16h逐渐恢复。结论：提示ST对小鼠SFO室管膜细胞有明显的损伤作用。     

An ultrastructural study of germ cells during ovarian differentiation in Torpedo marmorata

An ultrastructural investigation, performed on embryos, neonates, subadult and adult females, demonstrated that in Torpedo marmorata oogenesis occurs very early in life and continues, in its proliferative phase, also after birth. Clusters of early meiotic cells were already evident in the ovarian cortex of 6‐cm‐long embryos, as well as in the ovary of newborns and three‐month‐old young. Conversely, in the ovaries of subadult and adult females, all the germ cells present were organized into follicles, and no clusters of oogonia and early meiotic cells were generally found in the cortex, except for one adult female where clusters of germ cells not organized in follicles were found in the cortex. These data demonstrated that, in Torpedo marmorata, oogenesis is immediate, and, as oogonia persist after birth, more similar to that of mouse, monkey, rabbit, and ferret (Mauleon Arch Anat Microsc, 1967; 56:125–150; Byskov and Hoyer 1994 ) than to that of human, rat, pig, and guinea pig (Byskov and Hoyer 1994 ). Such a pattern is in agreement with the reproductive strategy of Torpedo, a scantly prolific species with low uterine fecundity. The presence of meiotic cells that are not organized in follicles in one adult female might be consistent with the large individual variability characterizing cartilaginous fishes. The possibility that such a character is typical of mature females should be rejected as oogonia and early meiotic cells were not found inside the totally sectioned gonads of subadult and adult females. Anat Rec 263:237–245, 2001. © 2001 Wiley‐Liss, Inc.     

Analysis using dual-colour fluorescencein situ hybridization of meiotic chromosome segregation in male mice heterozygous for a reciprocal translocation

Meiotic chromosome behaviour was investigated in male mice heterozygous for the translocation T(7;16)67H. At metaphase I, chain-of-four quadrivalents were present in approximately 80% of the spermatocytes; the bulk of remaining cells contained a ring quadrivalent, with only a few having either a trivalent plus univalent configuration or two bivalents. A low rate of non-disjunction, approximately 5%, was found through analysis of C-banded metaphase II spermatocytes. Using fluorescencein situ hybridization with differentially labelled whole chromosome paints, a wide array of segregation products were observed at metaphase II, depending on whether they arose from alternate, adjacent I, adjacent II orientation at metaphase I or were uninformative for alternative/adjacent I because of the presence of a chiasma in an interstitial pairing segment. Some 62% of the cells fell into this latter category, with only small proportions clearly arising through alternate (1.8%) or adjacent I (0.7%) orientations. Approximately 30% of the cells contained the products of adjacent II orientation. Consideration of the data suggested that most of these cells arose from metaphase I cells that contained a chain quadrivalent. Ring quadrivalents appeared predominantly to orientate in an alternate/adjacent I manner.for publication by J. S. (Pat) Heslop-Harrison     

Synaptonemal complexes of chains and rings in mice heterozygous for multiple Robertsonian translocations

Complex Robertsonian translocation heterozygosities in the mouse have been used to test different hypotheses regarding the correlation between male hybrid sterility and chromosomal abnormality. Synaptonemal complexes of meiotic super-chains and super-rings involving 15 to 18 metacentric chromosomes were studied in relation to spermatogenic histology. Both types of multivalents showed a characteristic pachytene pattern of alternating paired and non-paired segments. The amount of unpaired segments in rings was about 18% and in chains about 23% of the total length of multivalent chromosomes. The meiotic chains were associated with the proximal part of the X chromosomes in more than 60% of pachytene cells; a similar tight proximity of rings with X or Y chromosomes was never found. Complete arrest of germ cell maturation correlated with super-chains and inconspicuous testicular histology with super-rings. This demonstrates that an excessive amount of unpaired chromosomal axes does not leadper se to male infertility through gametogenic breakdown. On the contrary, the results clearly indicate spermatogenic impairment in this system of multimetacentric heterozygosity as a reflection of X chromosome super-chain interference.     

神经干细胞移植对小鼠脊髓损伤的影响

目的观察神经干细胞(NSCs)移植对脊髓损伤(SCI)小鼠功能恢复的影响。方法分离、培养、增殖和纯化E14-17d小鼠的NSCs,并通过免疫荧光法对其进行鉴定。将绿色荧光蛋白转基因小鼠的NSCs移植到小鼠脊髓损伤模型体内,术后进行行为学和病理学检测,观察小鼠功能恢复情况。运用改良Allen's法制备小鼠T10-T11脊髓损伤动物模型,动物分为假手术组(12只)、模型组(12只),治疗组(12只)和对照组6(12只)。治疗组每只小鼠自眶静脉注射NSCs悬液200μl(2×10个细胞),对照组只注射DMEM/F12培养基200μl。术后1d、3d、7d、14d、21d、28d和56d,进行BBB运动功能评分和病理学检测,观察植入区细胞生长变化情况。结果 BBB评分显示:治疗组明显高于对照组(<0.01),治疗组与假手术组相比没有明显差异(>0.05),说明NSCs移植后小鼠的行为学得到了明显改善,功能有所恢复。病理学检测发现,移植后NSCs不仅迁移到脊髓损伤区,而且与宿主细胞较好地整合。结论移植的E14-17d胚胎小鼠NSCs不仅可在脊髓损伤部位存活并和宿主细胞整合,而且可促进小鼠后肢运动功能恢复。     

染色体制备环节的量化及效果评价

目的量化控制染色体制备的9个环节,评价量化控制措施对染色体标本制作质量的影响。方法根据外周血淋巴细胞染色体制备的基本过程,对其中的9个环节严格定量控制,用同一份血样重复进行4次染色体标本制备,测量各次所获标本独立核型染色体的平均分布面积及9号染色体的平均长度,评价定量控制措施的实际效果。结果在9个环节得到严格控制后,4次重复实验均得到了丰富的可利用核型,可利用核型的平均分布面积并9号染色体的平均长度依次为:1246(113)、1143(108)、1217(117)和1167(118)mm2(mm)。结论我们的量化控制措施保证了日常染色体制备的稳定性和重复性。