Detection of Y-bearing spermatozoa by DNA-DNA in situ hybridisation

In situ hybridisation of a Y chromosome-specific DNA probe to preparations of decondensed spermatozoa revealed approximately 46.7% labelled spermatozoa among 3,900 scored. This is not significantly different from the 50% expected if only the Y chromosome-bearing spermatozoa are hybridised. Control hybridizations of Escherichia coli DNA and salmon testis DNA to decondensed sperm produced no significant labelling, whereas more than 99% of the spermatozoa were heavily labelled after hybridisation to total human DNA. These controls indicate that the methodology described in this paper renders the chromatin accessible for hybridisation and that the 50% hybridisation observed with the Y chromosome DNA probe was specific. In situ hybridisation with the Y probe therefore identifies the Y-bearing spermatozoa, and the protocol described should prove useful in evaluating methods of separating Y-bearing and X-bearing spermatozoa.     

泥鳅精子和卵子结构及受精过程的细胞学观察

研究旨在探讨泥鳅精子受精时限及为其人工繁殖提供基础资料。采用光镜、电镜技术对泥鳅（Misgurnus anguillicaudatus）精子、卵子和不同时间段的受精卵进行了观察。结果显示:泥鳅精子头部无顶体,主要为核占据,核凹窝较浅,中段具不对称的袖套,尾部轴丝为9+2结构,无侧鳍。卵子动物极卵膜仅有一受精孔,受精孔为深凹陷、短孔道型。在20-21℃水温条件下,授精后3s,精子开始穿过精孔管或已经进入卵子;授精后5-8s,形成精子星光;授精后70s,卵子处于第二次减数分裂后期;授精后6-8min,第二极体形成,等待排出;授精后20-25min,雌雄原核融合;授精后25-30min,受精卵进入第一次有丝分裂中期;授精后40-45min,第一次有丝分裂结束,二细胞形成。研究表明:成熟卵子精孔管内口径（2.2860.364）m,而精子头部直径与其接近,单精受精;入水后150s内可受精。     

The fusion of sperm cells and the function of male germ unit (MGU) of tobacco (Nicotiana tabacum L.)

 Sperm cells released from in vivo-in vitro grown pollen tubes of tobacco are associated in pairs and initially enclosed by the plasma membrane of the pollen tube. When the sperm cells are placed together, using glass microinjector needles, in an enzymatic solution, up to half undergo cellular fusion with subsequent nuclear fusion. The frequency of sperm cell fusion decreases with time during the elongation of the pollen tube, suggesting that mechanisms inhibiting self-fusion of sperm cells may develop as the pollen tube elongates through the style toward the ovule. This tendency may play an important role in inhibiting fusion of the two sperm cells inside the calcium-rich synergid where the male germ unit dissociates and sperm cells are transported to their target cells - the egg and central cell. Received: 25 August 1997 / Revision accepted 16 March 1998     

Arabinogalactan proteins at the cell surface ofBrassica sperm andLilium sperm and generative cells

Antibodies to arabinogalactan proteins were tested for binding to sperm cells ofBrassica campestris and to generative cells and sperm ofLilium longiflorum. Two monoclonal antibodies, JIM8 and JIM13, bound toBrassica sperm in pollen grains and pollen tubes and to isolated sperm. Sperm pairs retained within the vegetative cell inner plasma membrane fluoresced more brightly than single sperm, indicating that the vegetative cell inner plasma membrane that surrounds sperm pairs also contains arabinogalactan proteins. Isolated sperm pairs exhibited a uniform fluorescence while single sperm had patches of fluorescence. InLilium, isolated generative cells and single sperm cells bound antibodies in a patchy pattern. Antibodies to arabinogalactan proteins may be useful in describing the overall shape of sperm cells and for identifying sperm among other cell types.     

Attraction and transport of male gametes for fertilization

 Two capabilities are critical in attracting and transporting male gametes for fertilization: (1) the pollen tube must locate, enter and discharge its contents at the correct site within the female gametophyte, and (2) once inside the embryo sac, the non-motile male gametes must be transported to the egg and the central cells for double fertilization. This review summarizes current information about evidence for communication between embryo sac and pollen tube and the means by which the non-motile male gametes are transported from the aperture of the pollen tube to the site of gamete fusion. Received: 6 June 1996 / Revision accepted: 9 July 1996     

人精子抗原决定簇的末端单糖参与小鼠IgA单抗与抗原的结合

研究消化道免疫后获得的抗人精子单抗(特别是IgA单抗)的靶抗原分子性质,有助于了解哪一些抗原可以通过消化道免疫,刺激机体产生局部分泌性兔疫反应。这对于抗精子避孕疫苗研制中精子有效抗原和免疫避孕投药途径的选择,阐明精子局部免疫的发生机制和弄清免疫性不育病人的病因有一定的意义。本文对消化道免疫获得的33个抗精子IgA单抗,12个IgG和35个IgM单抗靶抗原的生化性质及末端单糖做了鉴定。免疫印迹的结果显示,IgA,IgM和IgG类单抗靶抗原的分子量范围分别为10—89,11—75和12—94 KDa。有12个单抗的靶抗原为非蛋白类的糖复合物,一个IgA单抗(A 22)的靶抗原为不含糖的蛋白。凝集素封闭和糖苷酶消化试验的结果显示,98.7%单抗的靶抗原分子末端含一种或几种糖。五种凝集素对IgA类单抗靶抗原的封闭效应均较强,表明IgA类单抗靶抗原的抗原决定簇含有岩藻糖,乙酰氨基葡萄糖,乙酰氨基半乳精,半乳糖和甘露糖等末端单糖者较多。IgG类单抗靶抗原的抗原决定簇则含有带岩藻糖,乙酰氨基半乳糖和α-甘露糖等末端单糖者较多。内切-β-半乳糖苷酶消化试验的结果表明,54.4%IgA类单抗的靶抗原为依赖半乳糖苷连接的糖肽化合物。这些结果表明,经消化道免疫能产生IgA及其它类别抗体的绝大多数人精子抗原为含多种类型末端单糖的膜表面分子。     

Reproductive success in large populations: empirical measures and theoretical predictions of fertilization in the sea biscuit Clypeaster rosaceus

For organisms that free-spawn gametes into the environment, sperm limitation can be a major determinant of reproductive success. Previous tests of sperm limitation have been restricted to very small experimental populations. Here we test and then use a fertilization model to explore sperm limitation in large populations. Predictions of the fertilization model are compared with measures of dye diffusion and in situ fertilization of the sea biscuit Clypeaster rosaceus (Linnaeus). The model could not be rejected in either test. Then this model was used to simulate large-scale spawning events in a natural population of C. rosaceus. The results of our simulations indicate that both population size and population density are important to fertilization over a very large range (2 to over 250000 individuals), but we also found an important interaction between population size and density. The importance of high density was great in small populations but negligible in large populations. This result may provide insight into why aggregation during spawning is not universally seen in nature. Overall, results indicate that sperm limitation can both constrain reproductive success and mediate social behaviors in a wide range in population sizes.     

猪精子体外获能与顶体反应的超微结构研究

用４种方法,检测了猪精子体外获得的效果。结果证明：高离子浓度的前培养液和猪镦泡液,具有促进获能过程的作用,实验还获得了获能后顶体反尖的一些重要的形态学变化资料,包括质膜的膨胀、断裂、顶体膨胀、顶体外膜内陷或原位局部囊泡化,质膜再全部丢失。顶体内膜直到与卵母细胞质膜融合,才发生可见的变化。受精过程无论体内或体外,都容易发生多精入卵,体外受精则更甚。在精子穿过卵丘细胞之间时,一方面开始进行顶体反应,另     

Bovine oviduct-specific glycoprotein: A potent factor for maintenance of viability and motility of bovine spermatozoa in vitro

In the cow, a specific glycoprotein-bovine oviduct-specific glycoprotein (BOGP)-is secreted by the epithelial cells of the oviduct at the follicular stage of the estrous cycle. In this study, we examined the effects of purified BOGP on the viability and motility of bovine spermatozoa in culture in vitro. Frozen-thawed bovine spermatozoa were incubated in modified Tyrode's solution (TALP) that contained purified BOGP (TALP-BOGP). In TALP-BOGP, both the viability and motility of bovine spermatozoa were more effectively maintained than in the control medium without any added protein. The increases in both the viability and motility of spermatozoa were dose-dependent. Spermatozoa were also incubated in TALP medium supplemented with bovine serum albumin, egg albumin, lactalbumin, or gastric mucin, and their viability and motility in these media were compared with that in TALP-BOGP. Both the viability and motility of spermatozoa were more effectively maintained in TALP-BOGP throughout a 12-hr incubation than in other media tested. An immunolabeling study demonstrated that a monoclonal antibody specific for BOGP reacted with the posterior region of the head, the middle portion, and the tail of spermatozoa that had been incubated with TALP-BOGP, suggesting that BOGP becomes specifically associated with particular regions of the spermatozoon. These results suggest that BOGP is a potent factor for maintenance of the viability and motility of sperm. On the basis of the present results, we also propose that BOGP may play an important role in sperm functions during the reproductive process. © 1995 wiley-Liss, Inc.     

精子表面凝集素受体细胞化学检测法研究──Ⅲ．标记－劈裂术及其方法学问题

本文应用标记劈裂术检测精子表面ＣｏｎＡ受体并主要从方法学上加以探讨。采用１８ｎｍ和６ｎｍ胶体金作标记物进行比较,表明６ｎｍ金标记效率高,与膜蛋白分子对应性好,并提示６ｎｍ胶体金似可对质膜ＰＦ面跨膜蛋白进行定性。文中对该方法的技术关键作了总结,表明标记－劈裂术是质膜大分子细胞化学研究的可靠的新技术。